Anti-il-2 antibodies and compositions and uses thereof

ABSTRACT

The present invention provides antibodies, or antigen-binding portions thereof, which specifically bind to IL-2 and reduce the affinity of IL-2 binding to IL-2Rα and IL-2Rβ. The invention further provides a method of obtaining such antibodies and nucleic acids encoding the same. The invention further relates to compositions and therapeutic methods for use of these antibodies for the treatment and/or prevention of autoimmune diseases, disorders or conditions and for immunosuppression, including, but not limited to, administering a complex comprising the antibody and IL-2.

PARTIES TO A JOINT RESEARCH AGREEMENT

The presently claimed invention was made by or on behalf of the belowlisted parties to a joint research agreement. The joint researchagreement was in effect on or before the date the claimed invention wasmade and the claimed invention was made as a result of activitiesundertaken within the scope of the joint research agreement. The partiesto the joint research agreement are PFIZER INC. and THE REGENTS OF THEUNIVERSITY OF CALIFORNIA.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/408,360, filed Oct. 14, 2016; and U.S. Provisional Application No.62/245,600, filed Oct. 23, 2015, the contents of each of whichapplications are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically as a text file in ASCII format and is herebyincorporated by reference in its entirety. Said text file, created onOct. 21, 2016, is named PCFC-993-101-SL.txt and is 106,553 bytes insize.

FIELD OF THE DISCLOSURE

The present disclosure relates to antibodies, e.g., full lengthantibodies and antigen-binding portions thereof that specifically bindinterleukin-2 (IL-2). The disclosure further relates to compositionscomprising antibodies to IL-2, and methods of using the antibodies as amedicament. The anti-IL-2 antibodies are useful for treating andpreventing autoimmune diseases, disorders and conditions and forimmunosuppression.

BACKGROUND OF THE DISCLOSURE

Interleukin-2 (IL-2) plays an important role in the immune response andis a potential target for treating diseases associated with the immuneresponse, such as autoimmune diseases, disorders and conditions and forimmunosuppression. There is a long-felt unmet need for noveltherapeutics to treat IL-2 mediated diseases, disorders, and conditions.The present disclosure meets these needs.

SUMMARY OF THE DISCLOSURE

The present inventors have generated new and advantageous anti-IL-2antibodies. In certain aspects, the disclosure relates to an isolatedantibody or an antigen-binding portion thereof that specifically bindshuman IL-2 (hIL-2), wherein the antibody reduces hIL-2 binding to IL-2receptor chains IL-2Rα and IL-2Rβ, and inhibits an activity in CD8⁺ Tcells to a higher degree than in regulatory T (Treg) cells.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds human IL-2(hIL-2), wherein the antibody binds helices A and C and the B-C loop ofhIL-2.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that competes for binding to human IL-2(hIL-2) with, or binds the same epitope of hIL-2 as, an antibodycomprising the amino acid sequences of SEQ ID NOs: 13 and 14.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds human IL-2(hIL-2), wherein the antibody reduces the binding affinity of hIL-2 toIL-2Rα by 1 to 199 fold. In certain embodiments, the antibody reducesthe binding affinity of hIL-2 to IL-2Rα by 10 fold.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds hIL-2, whereinthe antibody or portion reduces hIL-2 binding to IL-2Rα and IL-2Rβ, andinhibits STAT5 phosphorylation in CD8⁺ T cells to a higher degree thanin Treg cells.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds hIL-2, whereinthe antibody or portion reduces hIL-2 binding to IL-2Rα and IL-2Rβ, andincreases the ratio of Treg cells to CD8⁺ or CD4⁺ T cells or to NK cellsin the body, as measured in a peripheral blood mononuclear cell (PBMC)culture or reconstitution assay.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds hIL-2, whereinthe antibody or portion reduces hIL-2 binding to IL-2Rα and IL-2Rβ, andincreases expression of one or more of FOXP3, CD25, and Icos in Tregcells.

In certain aspects, the disclosure relates to an isolated antibody orantigen-binding portion, wherein the antibody or portion has a) an hIL-2binding off-rate of equal or greater than about 4.53×10⁻⁴ s⁻¹; and/or b)a binding affinity to hIL-2 of equal or greater than about 1.14×10⁻¹⁰ M.

In certain aspects, the disclosure relates to an isolated antibody orantigen-binding portion, wherein the antibody or portion comprises:

(a) a HCDR1 comprising SEQ ID NO: 73 (Kabat), 74 (Chothia), or 75(extended); a HCDR2 comprising SEQ ID NO: 76 (Kabat), or 77 (Chothia); aHCDR3 comprising SEQ ID NO: 78; a LCDR1 comprising SEQ ID NO: 79; aLCDR2 comprising SEQ ID NO: 80; and a LCDR3 comprising SEQ ID NO: 81;

(b) a HCDR1 comprising SEQ ID NO: 82 (Kabat), 83 (Chothia), or 84(extended); a HCDR2 comprising SEQ ID NO: 85 (Kabat), or 86 (Chothia); aHCDR3 comprising SEQ ID NO: 87; a LCDR1 comprising SEQ ID NO: 88; aLCDR2 comprising SEQ ID NO: 89; and a LCDR3 comprising SEQ ID NO: 90;

(c) a HCDR1 comprising SEQ ID NO: 91 (Kabat), 92 (Chothia), or 93(extended); a HCDR2 comprising SEQ ID NO: 94 (Kabat), or 95 (Chothia); aHCDR3 comprising SEQ ID NO: 96; a LCDR1 comprising SEQ ID NO: 97; aLCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;

(d) a HCDR1 comprising SEQ ID NO: 100 (Kabat), 101 (Chothia), or 102(extended); a HCDR2 comprising SEQ ID NO: 103 (Kabat), or 104 (Chothia);a HCDR3 comprising SEQ ID NO: 105; a LCDR1 comprising SEQ ID NO: 106; aLCDR2 comprising SEQ ID NO: 107; and a LCDR3 comprising SEQ ID NO: 108;

(e) a HCDR1 comprising SEQ ID NO: 109 (Kabat), 110 (Chothia), or 111(extended); a HCDR2 comprising SEQ ID NO: 112 (Kabat), or 113 (Chothia);a HCDR3 comprising SEQ ID NO: 114; a LCDR1 comprising SEQ ID NO: 115; aLCDR2 comprising SEQ ID NO: 116; and a LCDR3 comprising SEQ ID NO: 117;

(f) a HCDR1 comprising SEQ ID NO: 118 (Kabat), 119 (Chothia), or 120(extended); a HCDR2 comprising SEQ ID NO: 121 (Kabat), or 122 (Chothia);a HCDR3 comprising SEQ ID NO: 123; a LCDR1 comprising SEQ ID NO: 124; aLCDR2 comprising SEQ ID NO: 125; and a LCDR3 comprising SEQ ID NO: 126;

(g) a HCDR1 comprising SEQ ID NO: 127 (Kabat), 128 (Chothia), or 129(extended); a HCDR2 comprising SEQ ID NO: 130 (Kabat), or 131 (Chothia);a HCDR3 comprising SEQ ID NO: 132; a LCDR1 comprising SEQ ID NO: 133; aLCDR2 comprising SEQ ID NO: 134; and a LCDR3 comprising SEQ ID NO: 135;

(h) a HCDR1 comprising SEQ ID NO: 136 (Kabat), 137 (Chothia), or 138(extended); a HCDR2 comprising SEQ ID NO: 139 (Kabat), or 140 (Chothia);a HCDR3 comprising SEQ ID NO: 141; a LCDR1 comprising SEQ ID NO: 142; aLCDR2 comprising SEQ ID NO: 143; and a LCDR3 comprising SEQ ID NO: 144;

(i) a HCDR1 comprising SEQ ID NO: 145 (Kabat), 146 (Chothia), or147(extended); a HCDR2 comprising SEQ ID NO: 148 (Kabat), or 149(Chothia); a HCDR3 comprising SEQ ID NO: 150; a LCDR1 comprising SEQ IDNO: 151; a LCDR2 comprising SEQ ID NO: 152; and a LCDR3 comprising SEQID NO: 153;

(j) a HCDR1 comprising SEQ ID NO: 154 (Kabat), 155 (Chothia), or 156(extended); a HCDR2 comprising SEQ ID NO: 157 (Kabat), or 158 (Chothia);a HCDR3 comprising SEQ ID NO: 159; a LCDR1 comprising SEQ ID NO: 160; aLCDR2 comprising SEQ ID NO: 161; and a LCDR3 comprising SEQ ID NO: 162;

(k) a HCDR1 comprising SEQ ID NO: 163 (Kabat), 164 (Chothia), or 165(extended); a HCDR2 comprising SEQ ID NO: 166 (Kabat), or 167 (Chothia);a HCDR3 comprising SEQ ID NO: 168; a LCDR1 comprising SEQ ID NO: 169; aLCDR2 comprising SEQ ID NO: 170; and a LCDR3 comprising SEQ ID NO: 171;

(l) a HCDR1 comprising SEQ ID NO: 172 (Kabat), 173 (Chothia), or 174(extended); a HCDR2 comprising SEQ ID NO: 175 (Kabat), or 176 (Chothia);a HCDR3 comprising SEQ ID NO: 177; a LCDR1 comprising SEQ ID NO: 178; aLCDR2 comprising SEQ ID NO: 179; and a LCDR3 comprising SEQ ID NO: 180;

(m) a HCDR1 comprising SEQ ID NO: 181 (Kabat), 182 (Chothia), or 183(extended); a HCDR2 comprising SEQ ID NO: 184 (Kabat), or 185 (Chothia);a HCDR3 comprising SEQ ID NO: 186; a LCDR1 comprising SEQ ID NO: 187; aLCDR2 comprising SEQ ID NO: 188; and a LCDR3 comprising SEQ ID NO: 189;

(n) a HCDR1 comprising SEQ ID NO: 190 (Kabat), 191 (Chothia), or 192(extended); a HCDR2 comprising SEQ ID NO: 193 (Kabat), or 194 (Chothia);a HCDR3 comprising SEQ ID NO: 195; a LCDR1 comprising SEQ ID NO: 196; aLCDR2 comprising SEQ ID NO: 197; and a LCDR3 comprising SEQ ID NO: 198;

(o) a HCDR1 comprising SEQ ID NO: 199 (Kabat), 200 (Chothia), or 201(extended); a HCDR2 comprising SEQ ID NO: 202 (Kabat), or 203 (Chothia);a HCDR3 comprising SEQ ID NO: 204; a LCDR1 comprising SEQ ID NO: 205; aLCDR2 comprising SEQ ID NO: 206; and a LCDR3 comprising SEQ ID NO: 207;

(p) a HCDR1 comprising SEQ ID NO: 208 (Kabat), 209 (Chothia), or 210(extended); a HCDR2 comprising SEQ ID NO: 211 (Kabat), or 212 (Chothia);a HCDR3 comprising SEQ ID NO: 213; a LCDR1 comprising SEQ ID NO: 214; aLCDR2 comprising SEQ ID NO: 215; and a LCDR3 comprising SEQ ID NO: 216;or

(q) a HCDR1 comprising SEQ ID NO: 217; a HCDR2 comprising SEQ ID NO:218; a HCDR3 comprising SEQ ID NO: 219; a LCDR1 comprising SEQ ID NO:220; a LCDR2 comprising SEQ ID NO: 221; and a LCDR3 comprising SEQ IDNO: 222.

In some embodiments, the antibody or antigen-binding portion comprises aheavy chain variable domain (V_(H)) comprising: a) a heavy chaincomplementarity determining region (HCDR) 3 in SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 71 as shown in Table 7; or b)HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29 or 71 as shown in Table 7.

In some embodiments, the antibody or antigen-binding portion of thedisclosure comprises a light chain variable domain (V_(L)) comprising:a) a light chain complementarity determining region (LCDR) 3 in SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 72 asshown in Table 7; or b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, or 72 as shown in Table 7.

In some of the above embodiments, the antibody or antigen-bindingportion comprises: a) HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, or 71 as shown in Table 7; and/or b)LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, or 72 as shown in Table 7.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds humaninterleukin-2 (hIL-2), comprising a heavy chain variable domain (V_(H))comprising: a) an HCDR3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, or 71 as shown in Table 7; b) HCDR1-3 in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71, asshown in Table 7; or c) the amino acid sequence of SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds hIL-2,comprising a light chain variable domain (V_(L)) comprising: a) an LCDR3in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, or 72 as shown in Table 7; b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72 as shown in Table 7;or c) the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, or 72.

In some embodiments, the disclosure relates to an isolated antibody orantigen-binding portion whose V_(H) comprises a) an HCDR3 in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71, asshown in Table 7; b) HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, or 71, as shown in Table 7; or c) theamino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, or 71; and whose V_(L) comprises a) an LCDR3 in SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72as shown in Table 7; b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, or 72 as shown in Table 7; or c) theamino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, or 72.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds hIL-2, whereinthe antibody comprises the HCDR1-3 and LCDR1-3 amino acid sequences in:

-   -   SEQ ID NOs: 1 and 2,    -   SEQ ID NOs: 3 and 4,    -   SEQ ID NOs: 5 and 6,    -   SEQ ID NOs: 7 and 8,    -   SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 11 and 12,    -   SEQ ID NOs: 13 and 14,    -   SEQ ID NOs: 15 and 16,    -   SEQ ID NOs: 17 and 18,    -   SEQ ID NOs: 19 and 20,    -   SEQ ID NOs: 21 and 22,    -   SEQ ID NOs: 23 and 24,    -   SEQ ID NOs: 25 and 26,    -   SEQ ID NOs: 27 and 28,    -   SEQ ID NOs: 29 and 30,    -   SEQ ID NOs: 31 and 32, or    -   SEQ ID NOs: 71 and 72,        respectively, as shown in Table 7.

In certain aspects, the disclosure relates to an isolated antibody orantigen-binding portion thereof whose V_(H) and V_(L) comprises theamino acid sequences of

-   -   SEQ ID NOs: 1 and 2,    -   SEQ ID NOs: 3 and 4,    -   SEQ ID NOs: 5 and 6,    -   SEQ ID NOs: 7 and 8,    -   SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 11 and 12,    -   SEQ ID NOs: 13 and 14,    -   SEQ ID NOs: 15 and 16,    -   SEQ ID NOs: 17 and 18,    -   SEQ ID NOs: 19 and 20,    -   SEQ ID NOs: 21 and 22,    -   SEQ ID NOs: 23 and 24,    -   SEQ ID NOs: 25 and 26,    -   SEQ ID NOs: 27 and 28,    -   SEQ ID NOs: 29 and 30,    -   SEQ ID NOs: 31 and 32, or    -   SEQ ID NOs: 71 and 72, respectively.

In some embodiments of the disclosure, the isolated antibody is an IgG(e.g., IgG₁, IgG₂, IgG₃, or IgG₄). The antibody may comprise a heavychain constant region comprising the amino acid sequence of SEQ ID NO:33, and/or a light chain constant region comprising the amino acidsequence of SEQ ID NO: 34 or 35. In some of these embodiments, the heavychain C-terminal lysine is absent.

In certain aspects, the disclosure relates to an isolated antibody or anantigen-binding portion thereof that specifically binds hIL-2, whereinthe antibody binds to the same epitope as, or competes for binding toIL-2Rα and IL-2Rβ with, any of the above-described antibodies.

In certain aspects, the disclosure relates to an antibody orantigen-binding portion whose V_(H) and V_(L) amino acid sequences areat least 90% (e.g., 95%, 98%, or 99%) identical to the following aminoacid sequences, respectively:

-   -   SEQ ID NOs: 1 and 2,    -   SEQ ID NOs: 3 and 4,    -   SEQ ID NOs: 5 and 6,    -   SEQ ID NOs: 7 and 8,    -   SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 11 and 12,    -   SEQ ID NOs: 13 and 14,    -   SEQ ID NOs: 15 and 16,    -   SEQ ID NOs: 17 and 18,    -   SEQ ID NOs: 19 and 20,    -   SEQ ID NOs: 21 and 22,    -   SEQ ID NOs: 23 and 24,    -   SEQ ID NOs: 25 and 26,    -   SEQ ID NOs: 27 and 28,    -   SEQ ID NOs: 29 and 30, or    -   SEQ ID NOs: 31 and 32.

In some of the above-described embodiments of the disclosure, theantibody is a human antibody.

This disclosure also provides an isolated nucleic acid encoding theheavy chain, the light chain, or both, of an antibody or antigen-bindingportion of the disclosure.

In certain aspects, the isolated nucleic acid comprises: a) thenucleotide sequence of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, or 66; b) the nucleotide sequence of SEQ ID NO:37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67; or c)both a) and b). For example, the isolated nucleic acid may comprise thenucleotide sequences of:

-   -   SEQ ID NOs: 36 and 37,    -   SEQ ID NOs: 38 and 39,    -   SEQ ID NOs: 40 and 41,    -   SEQ ID NOs: 42 and 43,    -   SEQ ID NOs: 44 and 45,    -   SEQ ID NOs: 46 and 47,    -   SEQ ID NOs: 48 and 49,    -   SEQ ID NOs: 50 and 51,    -   SEQ ID NOs: 52 and 53,    -   SEQ ID NOs: 54 and 55,    -   SEQ ID NOs: 56 and 57,    -   SEQ ID NOs: 58 and 59,    -   SEQ ID NOs: 60 and 61,    -   SEQ ID NOs: 62 and 63,    -   SEQ ID NOs: 64 and 65, or    -   SEQ ID NOs: 66 and 67.

In certain aspects, the disclosure relates to a vector comprising one ormore of the above isolated nucleic acids. In other aspects, thedisclosure provides a host cell (e.g., mammalian cells such as NS0 cellsand CHO cells) comprising an isolated nucleic acid or vector encodingthe heavy chain, the light chain, or both, of an antibody orantigen-binding portion of the disclosure. The disclosure also providesa method of producing an antibody or an antigen-binding portion thereofthat specifically binds hIL-2, comprising: a) culturing a host cellunder conditions that allow said antibody or antigen-binding portion tobe expressed, wherein the host cell comprises nucleotide sequencescoding the heavy chain and light chain of the antibody orantigen-binding portion, and b) isolating said antibody orantigen-binding portion from the culture.

In certain aspects, the disclosure relates to a pharmaceuticalcomposition comprising an antibody or antigen-binding portion of thedisclosure and a pharmaceutically acceptable carrier or excipient.

In certain aspects, the disclosure relates to a method for treating aninflammatory condition such as an autoimmune disease or inducingimmunosuppression in a human subject in need thereof, comprisingadministering to the subject an effective amount of an antibody orantigen-binding portion of the disclosure or a pharmaceuticalcomposition of the disclosure. In some embodiments, the antibody isadministered in complex with IL-2. In related aspects, the disclosureprovides an antibody or antigen-binding portion of the disclosure or apharmaceutical composition of the disclosure for use in treating a humansubject having an inflammatory condition such as an autoimmune diseaseor in need of immunosuppression, wherein the antibody or portion isoptionally administered in complex with IL-2; and the use of an antibodyor antigen-binding portion of the disclosure in the manufacture of amedicament for treating an inflammatory condition such as an autoimmunedisease or inducing immunosuppression, wherein the antibody or portionis optionally administered in complex with IL-2.

The conditions that can be treated with the present compositions(including but not limited to antibody or antibody/IL-2 complexesdescribed herein) and methods include, but are not limited to:inflammatory skin diseases including psoriasis and dermatitis (e.g.,atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis;conditions associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); colitis; gastritis; respiratorydistress syndrome (including adult respiratory distress syndrome andARDS); dermatitis; meningitis; encephalitis; uveitis;glomerulonephritis; allergic conditions such as eczema and asthma andother conditions involving infiltration of T cells and chronicinflammatory responses; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus (e.g., Type I diabetes mellitus); multiple sclerosis; Reynaud'ssyndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren'ssyndrome; juvenile onset diabetes; and immune responses associated withacute and delayed hypersensitivity mediated by cytokines andT-lymphocytes typically found in tuberculosis, sarcoidosis,polymyositis, granulomatosis and vasculitis; Wegener's disease;pernicious anemia (Addison's disease); diseases involving leukocytediapedesis; central nervous system (CNS) inflammatory disorder; multipleorgan injury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; vitiligo; Reiter's disease;stiff-man syndrome; Behcet's disease; giant cell arteritis; immunecomplex nephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia andautoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmunehepatitis; autoimmune hemophilia; autoimmune lymphoproliferativesyndrome (ALPS); autoimmune uveoretinitis; Guillain-Barre syndrome;Goodpasture's syndrome; mixed connective tissue disease;autoimmune-associated infertility; polyarteritis nodosa; alopeciaareata; idiopathic myxedema; graft versus host disease; and musculardystrophy (Duchenne, Becker, Myotonic, Limb-girdle, Facioscapulohumeral,Congenital, Oculopharyngeal, Distal, and Emery-Dreifuss). In someembodiments, the condition that can be treated with the presentcompositions (including but not limited to antibody or antibody/IL-2complexes described herein) and methods is diabetes mellitus (e.g., TypeI diabetes mellitus). In some embodiments, the condition that can betreated with the present compositions (including but not limited toantibody or antibody/IL-2 complexes described herein) and methods isType I diabetes mellitus. In some embodiments, the condition that can betreated with the present compositions (including but not limited toantibody or antibody/IL-2 complexes described herein) and methods isjuvenile onset diabetes.

Other features and advantages of the disclosure will be apparent fromthe following detailed description, and from the Exemplary Embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor.

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 depicts antibody/IL-2 affinity and IL-2Rα and IL-2Rβ binding. Theresponse is reported as the binding to IL-2α and IL-2Rβ after 60 secondsas a percentage of the binding of two representative clones, d1C7 and16C3 in complex with IL-2, to IL-2Rα and IL-2Rβ respectively.

FIG. 2 depicts antibody/IL-2 affinity and IL-2Rα and IL-2Rβ binding.Both the parental and affinity matured clones showed complete inhibitionof the antibody/IL-2 complex binding to IL-2Rβ and a reduction in thebinding to IL-2Rα compared to the clone d1C7/IL-2 complex.

FIG. 3 depicts the phenotype of Tregs after IL-2:anti-IL-2 mAbtreatment. Treg population was gated on hCD45⁺ CD3⁺ CD4⁺ Helios' FoxP3⁺cells.

FIGS. 4A-C depict 16C3 (25 μg) and F5.1.11.02 (1, 5, and 25 μg) incomplex with hIL-2 increased Treg/CD4, Treg/CD8 and Treg/NK cell ratios.FIGS. 4D-F depict 16C3 (25 μg) and F5.1.11.02 (1 μg, 5 μg, and 25 μg) incomplex with hIL-2 increased Treg/CD4, Treg/CD8 and Treg/NK cell ratios.

FIGS. 5A-C depict that 16C3 (25 μg) and F5.1.11.02 (1, 5, and 25 μg) incomplex with hIL-2 increased the total number of CD4⁺, CD8⁺ cells andTregs in the spleen. FIGS. 5D-F depict that 16C3 (25 μg) and F5.1.11.02(1 μg, 5 μg, and 25 μg) in complex with hIL-2 increased the total numberof CD4⁺, CD8⁺ cells and Tregs in the spleen.

FIGS. 6A-C depict CD25, Icos and FoxP3 mean fluorescence intensity (MFI)on Tregs after antibody treatment.

FIGS. 7A-V depict pSTAT5 signaling in CD8⁺ effector T cells and Tregcells after antibody treatment. Curves represent treatment with 0.2ng/mL hIL-2, 10 ng/mL hIL-2, or 500 ng/mL hIL-2.

FIGS. 8A-F depict pSTAT5 signaling in CD8⁺ effector T cells and Tregcells after antibody treatment. Curves represent treatment with 0.5ng/mL hIL-2, 5 ng/mL hIL-2, 50 ng/mL hIL-2, or 500 ng/mL hIL-2.

FIGS. 9A-D depict pSTAT5 signaling in CD8⁺/CD25 high T cells, CD8⁺/CD25low T cells and Treg cells after antibody treatment. The x-axis is nMantibody. Curves represent treatment with 0.8 ng/mL hIL-2, 20 ng/mLhIL-2, or 500 ng/mL hIL-2.

FIGS. 10A-B depict pSTAT5 signaling in CD8⁺/CD25 high T cells, CD8⁺/CD25low T cells and Treg cells with varying concentrations of IL-2.

FIGS. 11A-H depict pSTAT5 signaling in CD8⁺ effector T cells and Tregcells after antibody F5.1.9, F5.1.9.5, and F5.1.11.04 treatment. Thex-axis is nM antibody. Curves represent treatment with 0.8 ng/mL hIL-2,20 ng/mL hIL-2, or 500 ng/mL hIL-2.

FIGS. 12A-B depict production of IL-2 after in vitro stimulation ofmouse splenocytes with PMA/lonomycin.

FIGS. 13A-B depict the percentage of activated (CD44⁺CD62L⁻) CD4⁺ orCD8⁺ T cells in hIL-2Tg, NOD, or NOD mIL-2+/−mice.

FIG. 14 depicts the cell surface expression of CD25 on Tregs from hIL-2Tg, NOD, or NOD mIL-2+/−mice.

FIG. 15 depicts the effect of F5.1.11.02:IL-2 complex on overallcellularity of the spleen at day 7.

FIGS. 16A-I depict the effect of F5.1.11.02:IL-2 complex on Tregpercentage in spleen, pLN and pancreas.

FIGS. 17A-F depict the effect of F5.1.11.02 (25 μg) in complex withhIL-2 on Treg/CD4 and Treg/CD8 ratios in spleen, pLN and pancreas.

FIGS. 18A-C depict the effect of F5.1.11.02:IL-2 complex (5 μg and 25 μgF5.1.11.02) on CD25 mean fluorescence intensity (MFI) on Tregs inspleen, pLN and pancreas.

FIGS. 19A-B depict the effect of 25 μg of F5.1.11.02 in complex withhIL-2 on the number of total splenocytes.

FIGS. 20A-F depict the effect of F5.1.11.02 antibody:IL-2 complex onTeff and Treg total cell number in the spleen. Treg population was gatedon hCD45⁺ CD3⁺ CD4⁺ Helios⁺ FoxP3⁺ cells.

FIGS. 21A-D depict the effect of F5.1.11.02 antibody:IL-2 complex onTreg/CD4 and Treg/CD8 ratios in the spleen. Treg population was gated onhCD45⁺ CD3⁺ CD4⁺ Helios⁺ FoxP3⁺ cells.

FIGS. 22A-H depict the effect of F5.1.11.02 antibody:IL-2 complextreatment on the proliferation of Tregs.

FIGS. 23A-H depict the effect of F5.1.11.02 antibody:IL-2 complextreatment on the proliferation of CD8 T cells.

FIGS. 24A-B depict treatment with both doses of the F5.1.11.02antibody:IL-2 complex induced an increase of CD25 mean fluorescenceintensity (MFI) on Tregs and on the CD8 population in the spleencompared to the isotype control.

FIGS. 25A-D depict a comparison between the effects of F5.1.11.02 andF5.1.11 on Treg and CD8 cell number.

FIGS. 26A-C depict a comparison between the effects of F5.1.11.02 andF5.1.11 on Treg/CD8 ratio.

FIGS. 27A-H depict the effect of antibodies on Treg proliferation atvarious doses.

FIGS. 28A-H depict the effect of antibodies on CD8 cell proliferation atvarious doses.

FIGS. 29A-B depict an equilibrium binding analysis for IL-2Rα binding toincreasing concentrations of IL-2 (A) and IL-2/F5.1.11 Fab (B).

FIG. 30 depicts the F5.1.11 Fab interaction with IL-2 via the lightchain (LC) CDR1 and CDR3 loops and the heavy chain (HC) CDR2 and CDR3loops.

FIG. 31 depicts the F5.1.11 Fab/IL-2 complex overlaid with theIL-2-receptor quaternary complex showing the binding sites of theF5.1.11 Fab and IL-2Rβ are overlapping (left hand panel). The IL-2conformation when bound to the F5.1.11 Fab shows a conformational changerelative to the receptor bound IL-2 resulting in an allostericmodulation of the IL-2Rα binding site (right hand panel).

FIG. 32 depicts the structure-based library design for non-human primatecross-reactivity. Alignment of the human (SEQ ID NO: 224; NCBI accessionnumber NP_000577.2) and cyno (SEQ ID NO: 225; predicted from referencegenome NCBI accession number NC_022276.1) IL-2 amino acid sequencesshows the IL-2 variable loop corresponds to the F5.1.11 Fab bindinginterface.

FIGS. 33A-B depict a comparison between the effects of F5.1.11.02 andF5.1.11 on splenocyte and hCD45⁺ cellularity.

FIGS. 34A-B depict a comparison between the effects of F5.1.11.02 andF5.1.11 on Treg and CD8 cell CD25⁺ expression.

FIGS. 35A-B depict F5.1.11.02 antibody:IL-2 complex effects on Treg,CD4, and CD8 total cell numbers five days after treatment.

FIGS. 36A-B depict F5.1.11.02 antibody:IL-2 complex effects on Treg/CD4and Treg/CD8 cell ratios five days after treatment.

FIGS. 37A-B depict the effects of treatment with F5.1.11.02antibody:IL-2 complex on CD25 mean fluorescent intensity (MFI) on Tregand CD8 populations compared to isotype control.

FIG. 38 depicts the effects of treatment with F5.1.11.02 antibody:IL-2complex on FoxP3 mean fluorescent intensity (MFI) on Tregs.

FIGS. 39A-B depict promotion of differential Treg expansion usingantibodies that bind different epitopes on IL-2 thereby inhibitingbinding to different receptor epitopes/binding domains: an IL-2Rαblocker (16C3.4), an IL2Rβ blocker (d1C7), and an IL2Rβ blocker thatalso reduced IL2's binding to IL-2Rα (F5.1.11.02). Statistical one-wayANOVA 16C3.4 vs d1c7 or 16C3.4 vs F5.1.11.02.

FIG. 40 depicts the effects of treatment with 16C3.4, d1C7, andF5.1.11.02 on CD25 mean fluorescent intensity (MFI) on Treg and CD8populations compared to isotype control.

FIG. 41 depicts diabetes remission by F5.1.11.02 antibody:IL-2 complex.

FIG. 42 depicts the effects of F5.1.11.02 antibody:IL-2 complex effecton Treg numbers and characteristics in the pancreas.

DETAILED DESCRIPTION OF THE DISCLOSURE

The inventors have invented new and advantageous anti-IL-2 antibodies orantigen-binding portions thereof that specifically bind to hIL-2 andreduce hIL-2 binding to IL-2Rα and IL-2Rβ. These antibodies and portionscan inhibit proliferation of non-Treg cells (including effector CD68⁺,non-Treg CD4⁺ and NK cells) more than they inhibit proliferation of Tregcells; increase Treg proliferation compared to an isotype controlantibody; and/or increase the ratio of Treg cells to non-Treg cells ormaintain Treg markers. The present antibodies are distinct from theIL-2Rα blocking antibodies described in International ApplicationPCT/US2015/011794 (now published as International Publication Number WO2015/109212 on Jul. 23, 2015), incorporated by reference in itsentirety, as the present antibodies reduce, but do not abrogate, hIL-2binding to IL-2Rα and block hIL-2 binding to IL-2Rβ.

The anti-IL-2 antibodies or antigen-binding portions thereof can be usedin the prevention, treatment, and/or amelioration of diseases, disordersor conditions caused by and/or associated with IL-2 activity. Suchdiseases, disorders or conditions include, but are not limited to, type1 diabetes, autoimmune diseases, Graft versus Host Disease and otherimmunologic diseases where Tregs mediate inflammation, among others, aswould be appreciated by one skilled in the art provided with theteachings disclosed herein.

General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001); Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow andLane Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1998); Coligan et al., ShortProtocols in Protein Science, John Wiley & Sons, NY (2003); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean,eds., Oxford University Press, 2000); Using antibodies: a laboratorymanual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press,1999); and The Antibodies (M. Zanetti and J. D. Capra, eds., HarwoodAcademic Publishers, 1995).

Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry,biochemistry, immunology, molecular biology, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

DEFINITIONS

The following terms, unless otherwise indicated, shall be understood tohave the following meanings: the term “isolated molecule” (where themolecule is, for example, a polypeptide, a polynucleotide, or anantibody or portion thereof) is a molecule that by virtue of its originor source of derivation (1) is not associated with naturally associatedcomponents that accompany it in its native state, (2) is substantiallyfree of other molecules from the same species (3) is expressed by a cellfrom a different species, or (4) does not occur in nature. Thus, amolecule that is chemically synthesized, or expressed in a cellularsystem different from the cell from which it naturally originates, willbe “isolated” from its naturally associated components. A molecule alsomay be rendered substantially free of naturally associated components byisolation, using purification techniques well known in the art. Moleculepurity or homogeneity may be assayed by a number of means well known inthe art. For example, the purity of a polypeptide sample may be assayedusing polyacrylamide gel electrophoresis and staining of the gel tovisualize the polypeptide using techniques well known in the art. Forcertain purposes, higher resolution may be provided by using HPLC orother means well known in the art for purification.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and in someembodiments, a substantially purified fraction is a composition whereinthe object species (e.g., a glycoprotein, including an antibody orreceptor) comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, in some embodiments,more than about 85%, 90%, 95%, and 99%. In some embodiments, the objectspecies is purified to essential homogeneity (contaminant species cannotbe detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. In certain embodiments a substantially pure material is atleast 50% pure (i.e., free from contaminants), in some embodiments, atleast 90% pure, in some embodiments, at least 95% pure, yet in someembodiments, at least 98% pure, and in some embodiments, at least 99%pure. These amounts are not meant to be limiting, and increments betweenthe recited percentages are specifically envisioned as part of thedisclosure.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable domain of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso, unless otherwise specified, any antigen-binding portion thereofthat competes with the intact antibody for specific binding, fusionproteins comprising an antigen-binding portion, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site. Antigen-binding portions include, for example, Fab,Fab′, F(ab′)₂, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelidantibodies), portions including complementarity determining regions(CDRs), single chain variable fragment antibodies (scFv), maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide. Depending on the antibody amino acid sequence of theconstant region of its heavy chains, immunoglobulins can be assigned todifferent classes. There are five major classes (i.e., isotypes) ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (subtypes), e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁ and IgA₂. The heavy-chain constant regions that correspond tothe different classes of immunoglobulins are called alpha, delta,epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The terms “antigen-binding portion” or “antigen-binding fragment” of anantibody (or simply “antibody portion”), as used interchangeably herein,refers to one or more portions of an antibody that retain the ability tospecifically bind to an antigen (e.g., IL-2). It has been shown that theantigen-binding function of an antibody can be performed by portions ofa full-length antibody. Examples of binding portions encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabportion, a monovalent portion consisting of the V_(L), V_(H), CL and CH₁domains; (ii) a F(ab′)2 portion, a bivalent portion comprising two Fabportions linked by a disulfide bridge at the hinge region; (iii) a Fdportion consisting of the V_(H) and CH₁ domains; (iv) a Fv portionconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb portion (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR), disulfide-linked Fvs (dsFv), andanti-idiotypic (anti-Id) antibodies and intrabodies. Furthermore,although the two domains of the Fv portion, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv)); see e.g., Bird et al.Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA85:5879-5883 (1988)). Such single chain antibodies are also intended tobe encompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in whichV_(H) and V_(L) domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448(1993); Poljak et al., 1994, Structure 2:1121-1123).

A “variable domain” of an antibody refers to the variable domain of theantibody light chain (V_(L)) or the variable domain of the antibodyheavy chain (V_(H)), either alone or in combination. As known in theart, the variable domains of the heavy and light chains each consist offour framework regions (FRs) connected by three complementaritydetermining regions (CDRs) also known as hypervariable regions, andcontribute to the formation of the antigen-binding site of antibodies.If variants of a subject variable domain are desired, particularly withsubstitution in amino acid residues outside a CDR (i.e., in theframework region), appropriate amino acid substitution, in someembodiments, conservative amino acid substitution, can be identified bycomparing the subject variable domain to the variable domains of otherantibodies which contain CDR1 and CDR2 sequences in the same canonicalclass as the subject variable domain (see, e.g., Chothia and Lesk, J.Mol. Biol. 196(4): 901-917, 1987).

In certain embodiments, definitive delineation of a CDR andidentification of residues comprising the binding site of an antibody isaccomplished by solving the structure of the antibody and/or solving thestructure of the antibody-ligand complex. In certain embodiments, thatcan be accomplished by any of a variety of techniques known to thoseskilled in the art, such as X-ray crystallography. In certainembodiments, various methods of analysis can be employed to identify orapproximate the CDRs. In certain embodiments, various methods ofanalysis can be employed to identify or approximate the CDRs. Examplesof such methods include, but are not limited to, the Kabat definition,the Chothia definition, the AbM definition, the contact definition, theconformational definition and the IMGT definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See, e.g.,Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17;Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See, e.g., Martin et al., 1989, Proc NatlAcad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for ModelingVariable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. TheAbM definition models the tertiary structure of an antibody from primarysequence using a combination of knowledge databases and ab initiomethods, such as those described by Samudrala et al., 1999, “Ab InitioProtein Structure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Thecontact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol.,5:732-45. In another approach, referred to herein as the “conformationaldefinition” of CDRs, the positions of the CDRs may be identified as theresidues that make enthalpic contributions to antigen binding. See,e.g., Makabe et al., 2008, Journal of Biological Chemistry,283:1156-1166. Still other CDR boundary definitions may not strictlyfollow one of the above approaches, but will nonetheless overlap with atleast a portion of the Kabat CDRs, although they may be shortened orlengthened in light of prediction or experimental findings thatparticular residues or groups of residues do not significantly impactantigen binding. As used herein, a CDR may refer to CDRs defined by anyapproach known in the art, including combinations of approaches. Themethods used herein may utilize CDRs defined according to any of theseapproaches. For any given embodiment containing more than one CDR, theCDRs may be defined in accordance with any of Kabat, Chothia, extended,AbM, contact, and/or conformational definitions. In certain embodiments,the extended CDR refers to all of the amino acid residues identified bythe Kabat and Chothia methods.

“Contact residue” as used herein with respect to an antibody or theantigen specifically bound thereby, refers to an amino acid residuepresent on an antibody/antigen comprising at least one heavy atom (i.e.,not hydrogen) that is within 4 Å or less of a heavy atom of an aminoacid residue present on the cognate antibody/antigen.

As known in the art, a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present disclosure may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature 348:552-554, for example. As used herein, “humanized”antibody refers to forms of non-human (e.g., murine) antibodies that arechimeric immunoglobulins, immunoglobulin chains, or portions thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. In some embodiments, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. The humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen bindingresidues.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable domain sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable domain sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody or vice versa. The term also encompasses an antibody comprisinga V region from one individual from one species (e.g., a first mouse)and a constant region from another individual from the same species(e.g., a second mouse).

The term “antigen (Ag)” refers to the molecular entity used forimmunization of an immunocompetent vertebrate to produce the antibody(Ab) that recognizes the Ag or to screen an expression library (e.g.,phage, yeast or ribosome display library, among others). Herein, Ag istermed more broadly and is generally intended to include targetmolecules that are specifically recognized by the Ab, thus includingportions or mimics of the molecule used in an immunization process forraising the Ab or in library screening for selecting the Ab. Thus, forantibodies of the disclosure binding to IL-2, full-length IL-2 frommammalian species (e.g., human, monkey, mouse and rat IL-2), includingmonomers and multimers, such as dimers, trimers, etc. thereof, as wellas truncated and other variants of IL-2, are referred to as an antigen.

Generally, the term “epitope” refers to the area or region of an antigento which an antibody specifically binds, i.e., an area or region inphysical contact with the antibody. Thus, the term “epitope” refers tothat portion of a molecule capable of being recognized by and bound byan antibody at one or more of the antibody's antigen-binding regions.Typically, an epitope is defined in the context of a molecularinteraction between an “antibody, or antigen-binding portion thereof”(Ab), and its corresponding antigen. Epitopes often consist of a surfacegrouping of molecules such as amino acids or sugar side chains and havespecific three-dimensional structural characteristics as well asspecific charge characteristics. In some embodiments, the epitope can bea protein epitope. Protein epitopes can be linear or conformational. Ina linear epitope, all of the points of interaction between the proteinand the interacting molecule (such as an antibody) occur linearly alongthe primary amino acid sequence of the protein. A “nonlinear epitope” or“conformational epitope” comprises noncontiguous polypeptides (or aminoacids) within the antigenic protein to which an antibody specific to theepitope binds. The term “antigenic epitope” as used herein, is definedas a portion of an antigen to which an antibody can specifically bind asdetermined by any method well known in the art, for example, byconventional immunoassays. Alternatively, during the discovery process,the generation and characterization of antibodies may elucidateinformation about desirable epitopes. From this information, it is thenpossible to competitively screen antibodies for binding to the sameepitope. An approach to achieve this is to conduct competition andcross-competition studies to find antibodies that compete orcross-compete with one another for binding to IL-2, e.g., the antibodiescompete for binding to the antigen.

As used herein, the terms “wild-type amino acid,” “wild-type IgG,”“wild-type antibody,” or “wild-type mAb,” refer to a sequence of aminoor nucleic acids that occurs naturally within a certain population(e.g., human, mouse, rats, cell, etc.).

As outlined elsewhere herein, certain positions of the antibody moleculecan be altered. By “position” as used herein is meant a location in thesequence of a protein. Positions may be numbered sequentially, oraccording to an established format, for example the EU index and Kabatindex can be used to number amino acid residues of an antibody. Forexample, position 297 is a position in the human antibody IgG1.Corresponding positions are determined as outlined above, generallythrough alignment with other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

The term “T regulatory cell” or “Treg” refers to a type of T cell thatmay be characterized by function or biological markers that are known toone of skill in the art (see Schmetterer et al., FASEB Vol. 26 (2012)).In certain embodiments, a Treg cell expresses one or more of thefollowing markers: TCR/CD3, CD4, CD25 and stabilized FOXP3 based ondemethylation of critical genomic elements of the FOXP3 locus.

The term “Treg sparing antibody” refers to an antibody that binds toIL-2 and detectably shifts the ratio of Treg:CD8 cells in favor of Tregcells. In some embodiments, the Treg sparing antibody inhibitsproliferation of CD8⁺ cells to a greater extent than it inhibits theproliferation of Tregs. In some embodiments, the Treg sparing antibodyincreases the Treg:CD8 cells ratio by at least two-fold.

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

An antibody that “preferentially binds” or “specifically binds” (usedinterchangeably herein) to an epitope is a term well understood in theart, and methods to determine such specific or preferential binding arealso well known in the art. A molecule is said to exhibit “specificbinding” or “preferential binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. Also, an antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration to that target in asample than it binds to other substances present in the sample. Forexample, an antibody that specifically or preferentially binds to anIL-2 epitope is an antibody that binds this epitope with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other IL-2 epitopes or non-IL-2 epitopes. It is also understoodby reading this definition, for example, that an antibody (or moiety orepitope) which specifically or preferentially binds to a first targetmay or may not specifically or preferentially bind to a second target.As such, “specific binding” or “preferential binding” does notnecessarily require (although it can include) exclusive binding.

A variety of assay formats may be used to select an antibody or peptidethat specifically binds a molecule of interest. For example, solid-phaseELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare,Piscataway, N.J.), KinExA, fluorescence-activated cell sorting (FACS),Octet™ (ForteBio, Inc., Menlo Park, Calif.) and Western blot analysisare among many assays that may be used to identify an antibody thatspecifically reacts with an antigen or a receptor, or ligand bindingportion thereof, that specifically binds with a cognate ligand orbinding partner. Typically, a specific or selective reaction will be atleast twice the background signal or noise, more typically more than 10times background, even more typically, more than 50 times background,more typically, more than 100 times background, yet more typically, morethan 500 times background, even more typically, more than 1000 timesbackground, and even more typically, more than 10,000 times background.Also, an antibody is said to “specifically bind” an antigen when theequilibrium dissociation constant (K_(D)) is ≦7 nM.

The term “binding affinity” is herein used as a measure of the strengthof a non-covalent interaction between two molecules, e.g., an antibodyor portion thereof and an antigen. The term “binding affinity” is usedto describe monovalent interactions (intrinsic activity). Bindingaffinity between two molecules may be quantified by determination of thedissociation constant (K_(D)). In turn, K_(D) can be determined bymeasurement of the kinetics of complex formation and dissociation using,e.g., the surface plasmon resonance (SPR) method (Biacore). The rateconstants corresponding to the association and the dissociation of amonovalent complex are referred to as the association rate constantsk_(a) (or k_(on)) and dissociation rate constant k_(d) (or k_(off)),respectively. K_(D) is related to k_(a) and k_(d) through the equationK_(D)=k_(d)/k_(a). The value of the dissociation constant can bedetermined directly by well-known methods, and can be computed even forcomplex mixtures by methods such as those set forth in Caceci et al.(1984, Byte 9: 340-362). For example, the K_(D) may be established usinga double-filter nitrocellulose filter binding assay such as thatdisclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard assays to evaluate the binding ability ofantibodies towards target antigens are known in the art, including forexample, ELISAs, Western blots, RIAs, and flow cytometry analysis, andother assays exemplified elsewhere herein. The binding kinetics andbinding affinity of the antibody also can be assessed by standard assaysknown in the art, such as Surface Plasmon Resonance (SPR), e.g. by usinga Biacore™ system, or KinExA.

In some embodiments, the antibody may bind to hIL-2 with a K_(D) ofabout 1.14×10⁻¹⁰ M or greater. For example, the antibody may bind tohIL-2 with a K_(D) of about 9×10⁻¹¹ M or greater. In some embodiments,the antibody may bind to hIL-2 with a K_(D) of about 8×10⁻¹¹ M orgreater. In some embodiments, the antibody may bind to hIL-2 with aK_(D) of about 7×10⁻¹¹ M or greater. In some embodiments, the antibodymay bind to hIL-2 with a K_(D) of about 6×10⁻¹¹ M or greater. In someembodiments, the antibody may bind to hIL-2 with a K_(D) of about5.00×10⁻¹¹ M or greater. These amounts are not meant to be limiting, andincrements between the recited values are specifically envisioned aspart of the disclosure. In some embodiments, the antibody may bind tohIL-2 with a k_(d) of about the same as the K_(D) of an antibody asshown in Table 3.

In some embodiments, the antibody may bind to hIL-2 with a k_(d) ofabout 4.53×10⁻⁴ s⁻¹ or greater. For example, the antibody may bind tohIL-2 with a k_(d) of about 3×10⁻⁴ s⁻¹ or greater. In some embodiments,the antibody may bind to hIL-2 with a k_(d) of about 1×10⁻⁴ s⁻¹ orgreater. In some embodiments, the antibody may bind to hIL-2 with ak_(d) of about 9×10⁻⁵ s⁻¹ or greater. In some embodiments, the antibodymay bind to hIL-2 with a k_(d) of about 7×10⁻⁵ s⁻¹ or greater. In someembodiments, the antibody may bind to hIL-2 with a k_(d) of about5.00×10⁻⁵ s⁻¹ or greater. These amounts are not meant to be limiting,and increments between the recited values are specifically envisioned aspart of the disclosure. In some embodiments, the antibody may bind tohIL-2 with a k_(d) of about the same as the k_(d) of an antibody asshown in Table 3.

A competitive binding assay can be conducted in which the binding of theantibody to the target antigen is compared to the binding of the targetby another ligand of that target, such as another antibody or a solublereceptor that otherwise binds the target. The concentration at which 50%inhibition occurs is known as the K_(i). Under ideal conditions, theK_(i) is equivalent to K_(D). The K_(i) value will never be less thanthe K_(D), so measurement of K_(i) can conveniently be substituted toprovide an upper limit for K_(D).

Following the above definition, binding affinities associated withdifferent molecular interactions, e.g., comparison of the bindingaffinity of different antibodies for a given antigen, may be compared bycomparison of the K_(D) values for the individual antibody/antigencomplexes. Similarly, the specificity of an interaction may be assessedby determination and comparison of the K_(D) value for the interactionof interest, e.g., a specific interaction between an antibody and anantigen, with the K_(D) value of an interaction not of interest, e.g., acontrol antibody known not to bind IL-2.

An antibody that specifically binds its target may bind its target witha high affinity, that is, exhibiting a low K_(D) as discussed above, andmay bind to other, non-target molecules with a lower affinity. Forexample, the antibody may bind to non-target molecules with a K_(D) of1×10⁻⁶ M or more, in some embodiments, 1×10⁻⁵ M or more, in someembodiments, 1×10⁻⁴ M or more, in some embodiments, 1×10⁻³ M or more, insome embodiments, 1×10⁻² M or more. An antibody of the disclosure is insome embodiments capable of binding to its target with an affinity thatis at least two-fold, 10-fold, 50-fold, 100-fold 200-fold, 500-fold,1,000-fold or 10,000-fold or greater than its affinity for binding toanother non-IL-2 molecule. These amounts are not meant to be limiting,and increments between the recited values are specifically envisioned aspart of the disclosure.

An antibody:IL-2 “complex” as the term is used herein, refers to acomplex comprising at least one antibody, or antigen-binding portionthereof, of the present disclosure that specifically binds IL-2 and atleast one IL-2 cytokine molecule. The complex comprises an antibody andan IL-2 molecule that are associated by covalent, non-covalent, or anyother force. Preferably, the antibody and IL-2 may remain associated asa complex even after the complex is administered. It is understood thatthe antibody and IL-2 will form a complex based on, among othervariables, the KD value for the binding interaction between them.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected and/or transformed in vivo with a polynucleotide of thisdisclosure.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain may vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as described in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant domains, CH2 and CH3. Asis known in the art, an Fc region can be present in dimer or monomericform.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g., B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g., an antibody variable domain orantigen-binding portion thereof) and can be assessed using variousassays known in the art for evaluating such antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. In some embodiments, the variant Fc region has atleast one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g., from about oneto about ten amino acid substitutions, and in some embodiments, fromabout one to about five amino acid substitutions in a native sequence Fcregion or in the Fc region of the parent polypeptide. The variant Fcregion herein will in some embodiments possess at least about 80%sequence identity with a native sequence Fc region and/or with an Fcregion of a parent polypeptide, and in some embodiments, at least about90% sequence identity therewith, in some embodiments, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99% sequence identity therewith. These amounts are not meantto be limiting, and increments between the recited percentages arespecifically envisioned as part of the disclosure.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. In some embodiments, the FcR is anative sequence human FcR. Moreover, in some embodiments, FcR is onewhich binds an IgG antibody (a gamma receptor) and includes receptors ofthe FcγRI, FcγRII, and FcγRIII subclasses, including allelic variantsand alternatively spliced forms of these receptors. FcγRII receptorsinclude FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. FcRs are reviewed inRavetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al.,1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin.Med., 126:330-41. “FcR” also includes the neonatal receptor, FcRn, whichis responsible for the transfer of maternal IgGs to the fetus (Guyer etal., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol.,24:249).

As used herein, a first antibody is said to compete with a secondantibody for binding to an antigen (or an epitope) when the firstantibody's presence detectably decreases the binding of the secondantibody to the antigen (or the second antibody's epitope). Theconverse, where the binding of the first antibody to the antigen (or itsepitope) is also detectably decreased in the presence of the secondantibody, can, but need not, be true. However, where each antibodydetectably inhibits the binding of the other antibody to a commonantigen, whether to the same or a different extent, the antibodies aresaid to “cross-compete” with each other for binding of that antigen.Both competing and cross-competing antibodies are encompassed by thepresent disclosure. Regardless of the mechanism by which suchcompetition or cross-competition occurs (e.g., steric hindrance,conformational change, or binding to a common epitope or portion(s)thereof), the skilled artisan would appreciate, based upon the teachingsprovided herein, that such competing and/or cross-competing antibodiesare encompassed and can be useful for the methods disclosed herein.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improved survival rate (reduced mortality), reduction ininflammation, reduction in the amount of tissue fibrosis, improvement inthe appearance of the disease lesions, limitation of the pathologicallesions to focal sites, decreased extent of damage from the disease,decreased duration of the disease, and/or reduction in the number,extent, or duration of symptoms related to the disease. The termincludes the administration of the compounds or agents of the presentdisclosure to prevent or delay the onset of the symptoms, complications,or biochemical indicia of a disease, alleviating the symptoms orarresting or inhibiting further development of the disease, condition,or disorder. Treatment may be prophylactic (to prevent or delay theonset of the disease, or to prevent the manifestation of clinical orsubclinical symptoms thereof) or therapeutic suppression or alleviationof symptoms after the manifestation of the disease.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an IL-2 antibody. “Ameliorating” alsoincludes shortening or reduction in duration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toaffect any one or more beneficial or desired results. In more specificaspects, an effective amount prevents, alleviates or amelioratessymptoms of disease or infection, and/or prolongs the survival of thesubject being treated. For prophylactic use, beneficial or desiredresults include eliminating or reducing the risk, lessening theseverity, or delaying the outset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as reducing one or more symptomsof an IL-2 mediated disease, disorder or condition or an IL-2 deficiencydisease, disorder or condition, decreasing the dose of other medicationsrequired to treat the disease, enhancing the effect of anothermedication, and/or delaying the progression of the disease of patients.An effective dosage can be administered in one or more administrations.For purposes of this disclosure, an effective dosage of drug, compound,or pharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “vector” means a construct, which is capable ofdelivering, and, in some embodiments, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. In someembodiments, diluents for aerosol or parenteral administration arephosphate buffered saline (PBS) or normal (0.9%) saline. Compositionscomprising such carriers are formulated by well known conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; andRemington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000).

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g. 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Any example(s) following the term “e.g.” or “forexample” is not meant to be exhaustive or limiting.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the disclosure are described in terms ofa Markush group or other grouping of alternatives, the presentdisclosure encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group, but also the main group absent one or more of the groupmembers. The present disclosure also envisages the explicit exclusion ofone or more of any of the group members in the claimed disclosure.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present disclosure. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Overview

Interleukin-2 (IL-2) is a four-helix bundle, type I cytokine thatfunctions as a growth factor for a wide range of leukocytes, including Tcells and natural killer (NK) cells. Considerable effort has beeninvested in the study of IL-2 as a therapeutic target for a variety ofimmune disorders ranging from AIDS to cancer. Recombinant human IL-2(Proleukin®) is used at high doses to treat metastatic melanoma andrenal cell carcinoma. However, only a small subset of patients (5-10%)experience long-term survival from such treatment. Adverse effects ofhigh-dose IL-2 therapy, ranging from flu-like symptoms tolife-threatening vascular leak syndrome and pulmonary edema, havegreatly limited its use. Importantly, IL-2 treatment has unpredictablebiologic effects.

IL-2 mediates its effects by binding to a complex receptor comprised of3 chains CD25 (IL-2Rα), CD122 (IL-2Rβ), and common gamma chain (γ_(c),)such that the receptor is termed IL-2Rαβγ. The three chains aredifferentially expressed with the receptor trimer exhibiting the highestaffinity. Individually, each of the three receptor chains in thequaternary complex bind to hIL-2 with low affinity, with hIL-2Rα havingthe strongest relative affinity (K_(D)˜10 nM). Expression of hIL-2Rαincreases the on-rate of hIL-2 for T cells and the three receptorscooperatively bind to hIL-2 with K_(D)˜10 μM. Upon capture by IL-2Rα,IL-2 is presented to IL-2Rβ, and then γ_(c), possibly in a pre-formedreceptor dimer, to form the quaternary signaling complex. While hIL-2alone binds hIL-2Rβ with low affinity (K_(D)˜150-300 nM), thehIL-2/hIL-2Rα complex binds to hIL-2Rβ with higher affinity (K_(D)˜60nM), yet there is no direct contact between IL-2Rα and IL-2Rβ in thequaternary receptor complex. Recent studies suggest that wild-type IL-2exists in a “quiescent” form that is induced into a structurally-alteredconformation that represents the high affinity form which engagesIL-2Rα.

Attempts have been made to engineer or modify IL-2 to improve itstherapeutic potential by modifying its ability to selectively targeteither T effector cells (Teff) or T regulatory cells (Treg).Interestingly, there have been a number of approaches to more effectiveand directive IL-2s. In mouse models, which are not directly analogousto humans, Boyman and colleagues have demonstrated that, in somecircumstances, a rat anti-mouse IL-2 mAb (JES6-1) can be complexed withwild type IL-2 and used to preferentially enhance T_(REG) populations(Boyman et al., 2006, Science 311:1924-1927; and International PatentPublication No. WO 2007/095643). Although the mechanistic basis for thiseffect has not been elucidated, it may be, without wishing to be boundby any particular theory, that IL-2 in complex with certain antibodiesis “fixed” in a conformation that selectively triggers discriminatorysignals resulting in selective expansion of individual T_(REG) orT_(EFF) cell subsets. In addition, several efforts have been focused ondeveloping IL-2 mutant proteins which enhance activation of CD25⁺ Tcells and minimize activation of CD25⁻ NK cells by increasing IL-2binding to the trimolecular IL-2R complex CD25. In this regard, a mutantIL-2 has been made by Bayer with a significantly lower affinity for theIL-2Rβγ_(c) with the expectation that treatment with this variant IL-2may not activate NK or memory CD8⁺ T cells. However, both the IL-2mutant and IL-2/anti-IL-2 antibody complex approaches described above donot take into account the potential inability of a drug to alterendogenous wild type IL-2 that is being produced during the inflammatoryT cell response. Moreover, any therapy with IL-2 variants that induceendogenous IL-2 (a demonstrated feedback mechanism) can result in wildtype IL-2 effects in vivo that may not have been observed in vitro.

Therefore, although IL-2 was originally developed as an immunestimulatory agent due to its ability to enhance effector T cell(T_(EFF)) and NK function, it is now believed that the primary functionof IL-2 is NOT activation of immunity but rather the generation andsurvival of T_(REG), which function to inhibit immune responses andprevent autoimmune disease. There has been an increased understandingusing animal models, including IL-2 and IL-2 receptor (IL-2R)-deficientmice, that IL-2 plays a crucial role in peripheral immune tolerancemediated by T_(REG) cells. Studies have shown that low dose IL-2 therapypreferentially activates T_(REG) due to its constitutive expression ofthe high affinity IL-2R. More specifically, an increasing number ofanimal studies have demonstrated that T_(REG) can suppress GVHD andautoimmunity. Further, this potential suppressive role has beendemonstrated in humans in two recent studies, one in GVHD and the otherin autoimmune vasculitis, where short term low dose therapy led toamelioration of disease in some individuals. Thus, there is a long-feltneed for IL-2-based therapeutics to selectively activate the tolerogenicversus effector immune response, i.e., therapies designed to tip theT_(REG): T_(EFF) balance towards T_(REG), thereby treating a wide rangeof diseases. The present disclosure meets that need.

IL-2 Antibodies

The present invention relates to antibodies that specifically bind toIL-2, i.e., they bind to IL-2 but they do not detectably bind, or bindat a lower affinity, to other molecules. In some embodiments, theantibodies specifically bind to human IL-2. In some embodiments, theantibodies specifically bind helices A and C and the B-C loop of hIL-2.In some embodiments, the antibodies specifically bind helix A of hIL-2.In some embodiments, the antibodies specifically bind helix C of hIL-2.In some embodiments, the antibodies specifically bind helices A and C ofhIL-2. In some embodiments, the antibodies specifically bind the B-Cloop of hIL-2. In some embodiments, the antibodies compete for bindingto human IL-2 (hIL-2), with, or bind the same epitope of hIL-2 as, anantibody comprising the amino acid sequences of SEQ ID NOs: 13, 14, 126,127, 128, 129, 130, 131, 132, 133, and 134. In some embodiments, theantibodies specifically bind human IL-2 (hIL-2), and reduce the bindingaffinity of hIL-2 to IL-2Rα by about 1 fold to about 199 fold. In someembodiments, the antibodies reduce the binding affinity of hIL-2 toIL-2Rα by about 10 fold. In some embodiments, the antibodies reduce thebinding affinity of hIL-2 to IL-2Rα by about 2, 10, 25, 50, 75, 100,125, 150, or 175 fold.

In some embodiments, the IL-2 antibody reduces IL-2 binding to IL-2Rαand IL-2Rβ, and does not inhibit the activity of regulatory T (Treg)cells. For example, in some embodiments, the IL-2 antibody blocks IL-2binding to IL-2Rβ, and reduces the affinity of hIL-2 binding to IL-2Rα.In some embodiments, the IL-2 antibody reduces IL-2 binding to IL-2Rα byabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 99%. In some embodiments, the IL-2 antibody reduces IL-2binding to IL-2Rβ by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or99%. In some embodiments, the IL-2 antibody completely blocks IL-2binding to IL-2Rβ. In particular, the disclosure relates to antibodiesthat specifically bind to IL-2 and further, antibodies that inhibitproliferation of non-Treg cells (including effector CD8⁺, non-Treg CD4⁺and NK cells) more than they inhibit proliferation of Treg cells orincrease Treg proliferation compared to an isotype control antibody orincrease the ratio of Treg cells to non-Treg cells or maintain Tregmarkers or a combination thereof. In some embodiments, the antibodiesinhibit proliferation of CD8⁺, non-Treg CD4⁺ or NK cells at least 2-foldmore than said antibody inhibits the proliferation of Tregs. In someembodiments, the antibodies inhibit proliferation of CD8⁺, non-Treg CD4⁺or NK cells at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20-fold more than said antibody inhibits the proliferationof Tregs. In some embodiments, the antibodies reduce IL-2 binding toIL-2Rα and IL-2Rβ and increase the ratio of T regulatory cells (Tregs)to CD8⁺, non-Treg CD4⁺ or NK cells in a peripheral blood mononuclearcell (PBMC) culture or reconstitution assay. In some embodiments, theratio is increased at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20-fold or greater. In some embodiments, theantibody increases Tregs:CD8⁺ cells ratio by two (2) fold or greater,and may reflect increased Treg proliferation. In some embodiments, theantibodies enhance T regulatory cell (Treg) proliferation greater thanan isotype control when IL-2 is limiting, for example, at aconcentration of less than 1 nM in vitro. In some embodiments, theantibodies inhibit proliferation of CD8⁺ cells. In some embodiments, theantibodies reduce IL-2 binding to IL-2Rα and IL-2Rβ and maintainexpression of one or more FOXP3, CD25, and Icos in Treg cells. In someembodiments, the antibodies reduce IL-2 binding to IL-2Rα and IL-2Rβ andincrease expression of one or more FOXP3, CD25, and Icos in Treg cells.In some embodiments, an IL-2 antibody of the disclosure has at least oneof these features, and in some embodiments, the antibody has two or moreof these features. In some embodiments, the antibodies have all of thefeatures.

In some embodiments, the antibody, or antigen-binding portion thereof,that specifically binds IL-2, reduces IL-2 binding to IL-2Rα and IL-2Rβ,and inhibits STAT5 phosphorylation in CD8⁺ T cells to a higher degreethan in regulatory T (Treg) cells. In some embodiments, the IL-2antibody, or antigen-binding portion maintains STAT5 phosphorylation inTregs by greater than 50%. In some embodiments, the IL-2 antibody, orantigen-binding portion maintains STAT5 phosphorylation in Tregs bygreater than 60%. In some embodiments, the IL-2 antibody, orantigen-binding portion maintains STAT5 phosphorylation in Tregs bygreater than 70%. In some embodiments, the IL-2 antibody, orantigen-binding portion maintains STAT5 phosphorylation in Tregs bygreater than 80%. In some embodiments, the IL-2 antibody, orantigen-binding portion maintains STAT5 phosphorylation in Tregs bygreater than 90%. In some embodiments, the antibodies specifically bindto hIL-2. These amounts are not meant to be limiting, and incrementsbetween the recited percentages are specifically envisioned as part ofthe disclosure.

The disclosure also relates to compositions comprising such antibodiesas well as uses for such antibodies, including therapeutic andpharmaceutical uses.

By the term “IL-2” is meant any naturally occurring form of IL-2,whether monomeric or multimeric, including dimers, trimers, etc., whichmay be derived from any suitable organism. As used herein, “IL-2” refersto a mammalian IL-2, such as human, rat or mouse, as well as non-humanprimate, bovine, ovine, or porcine IL-2. In some embodiments, the IL-2is human (see, e.g., Genbank Accession Number P60568) or “hIL-2.” TheIL-2 can also be cynomolgus monkey IL-2 (see, e.g., Genbank AccessionNumber Q29615). The term “IL-2” also encompasses portions, variants,isoforms, and other homologs of such IL-2 molecules. Variant IL-2molecules will generally be characterized by having the same type ofactivity as naturally occurring IL-2, such as the ability to bind IL-2receptor, and the ability to induce receptor-mediated activity.

The IL-2 may comprise one or more, two or more, three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more, twelve or more or fifteen or more surface accessibleresidues of IL-2. Where the IL-2 comprises a homomultimeric form ofIL-2, the target may comprise one or more, two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, twelve or more, or fifteen or more surfaceaccessible residues of a first subunit of IL-2, and one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, ten or more, twelve or more, orfifteen or more surface accessible residues of a second subunit of IL-2.

The target molecule may comprise a known epitope from IL-2. The targetmolecule may comprise a known epitope from hIL-2. In some embodiments,the target may comprise helix A of hIL-2. In some embodiments, thetarget may comprise helix C of hIL-2. In some embodiments, the targetmay comprise helices A and C of hIL-2. In some embodiments, the targetmay comprise the B-C loop of hIL-2. In some embodiments, the target maycomprise helices A and C and the B-C loop of hIL-2.

In one embodiment, the disclosure provides any of the following, orcompositions (including pharmaceutical compositions) comprising, anantibody having a light chain sequence, or a portion thereof, and aheavy chain, or a portion thereof, derived from any of the followingantibodies: F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01,F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07,F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7. Antibody F5.1.11 is alsoreferred to as antibody F5111, 5.1.11, 5111. These terms areinterchangeable. Variants of this parental antibody may be referred toby this nomenclature with an additional number, e.g., antibody F5111.2,5.1.11.2, F5.1.11.02, or 5111.2; or antibody F5.1.11.01, F5111.1,5.1.11.1, or 5111.1.

The antibodies useful in the present disclosure can encompass monoclonalantibodies, polyclonal antibodies, antibody portions (e.g., Fab, Fab′,F(ab′)₂, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody),humanized antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies). In someembodiments, the IL-2 antibody is a monoclonal antibody. In someembodiments, the antibody is a human or humanized antibody.

The IL-2 antibodies of the disclosure may be made by any method known inthe art. General techniques for production of human and mouse antibodiesare known in the art and/or are described herein.

IL-2 antibodies can be identified or characterized using methods knownin the art, whereby reduction, amelioration, or neutralization of IL-2activity is detected and/or measured, for example, pAKT and/or pSTAT5.In some embodiments, an IL-2 antibody is identified by incubating acandidate agent (e.g., IL-2) with IL-2 receptor and monitoring bindingand/or attendant reduction or inhibition of a biological activity ofIL-2. In some embodiments, hIL-2 antibodies can be identified orcharacterized using methods known in the art, whereby reduction,amelioration, or neutralization of hIL-2 activity is detected and/ormeasured, for example, pAKT and/or pSTAT5. In some embodiments, a hIL-2antibody is identified by incubating a candidate agent (e.g., hIL-2)with hIL-2 receptor and monitoring binding and/or attendant reduction orinhibition of a biological activity of hIL-2. The binding assay may beperformed with, e.g., purified IL-2 polypeptide(s), or with cellsnaturally expressing various receptors, or transfected to express, IL-2receptor. In one embodiment, the binding assay is a competitive bindingassay, where the ability of a candidate antibody to compete with a knownIL-2 antibody for IL-2 binding is evaluated. The assay may be performedin various formats, including the ELISA format. In some embodiments, anIL-2 antibody is identified by incubating a candidate antibody with IL-2and monitoring binding.

Following initial identification, the activity of a candidate IL-2antibody can be further confirmed and refined by bioassays, known totest the targeted biological activities. In some embodiments, an invitro cell assay is used to further characterize a candidate IL-2antibody. For example, bioassays can be used to screen candidatesdirectly. Some of the methods for identifying and characterizing IL-2antibody are described in detail in the Examples.

IL-2 antibodies may be characterized using methods well known in theart. For example, one method is to identify the epitope to which itbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan IL-2 antibody binds. Epitope mapping is commercially available fromvarious sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PHLelystad, The Netherlands). The epitope can be a linear epitope, i.e.,contained in a single stretch of amino acids, or a conformationalepitope formed by a three-dimensional interaction of amino acids thatmay not necessarily be contained in a single stretch. Peptides ofvarying lengths (e.g., at least 4-6 amino acids long) can be isolated orsynthesized (e.g., recombinantly) and used for binding assays with IL-2antibody. In another example, the epitope to which the IL-2 antibodybinds can be determined in a systematic screening by using overlappingpeptides derived from the IL-2 sequence and determining binding by theantibody. According to the gene fragment expression assays, the openreading frame encoding IL-2 can be fragmented either randomly or byspecific genetic constructions and the reactivity of the expressedfragments of IL-2 with the antibody to be tested is determined. The genefragments may, for example, be produced by PCR and then transcribed andtranslated into protein in vitro, in the presence of radioactive aminoacids. The binding of the antibody to the radioactively labeled IL-2fragments is then determined by immunoprecipitation and gelelectrophoresis. Certain epitopes can also be identified by using largelibraries of random peptide sequences displayed on the surface of phageparticles (phage libraries) or yeast (yeast display). Alternatively, adefined library of overlapping peptide fragments can be tested forbinding to the test antibody in simple binding assays. In an additionalexample, mutagenesis of an antigen, domain swapping experiments andalanine scanning mutagenesis can be performed to identify residuesrequired, sufficient, and/or necessary for epitope binding. For example,alanine scanning mutagenesis experiments can be performed using a mutantIL-2 in which various residues of the IL-2 polypeptide have beenreplaced with alanine. By assessing binding of the antibody to themutant IL-2, the importance of the particular IL-2 residues to antibodybinding can be assessed.

Yet another method which can be used to characterize an IL-2 antibody isto use competition assays with other antibodies known to bind to thesame antigen, i.e., various fragments on IL-2, to determine if the IL-2antibody binds to the same epitope as other antibodies. Competitionassays are well known to those of skill in the art.

Further, the epitope for a given antibody/antigen binding pair can bedefined and characterized at different levels of detail using a varietyof experimental and computational epitope mapping methods. Theexperimental methods include mutagenesis, X-ray crystallography, NuclearMagnetic Resonance (NMR) spectroscopy, hydrogen/deuterium exchange MassSpectrometry (H/D-MS) and various competition binding methods well-knownin the art. As each method relies on a unique principle, the descriptionof an epitope is intimately linked to the method by which it has beendetermined. Thus, the epitope for a given antibody/antigen pair will bedefined differently depending on the epitope mapping method employed.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be defined by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level the epitope can be characterized by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At afurther less detailed level the epitope can be characterized by theamino acid residues that it comprises as defined by a specificcriterion, e.g., by distance between atoms (e.g., heavy, i.e.,non-hydrogen atoms) in the Ab and the Ag. At a further less detailedlevel the epitope can be characterized through function, e.g. bycompetition binding with other Abs. The epitope can also be defined moregenerically as comprising amino acid residues for which substitution byanother amino acid will alter the characteristics of the interactionbetween the Ab and Ag (e.g. using alanine scanning).

From the fact that descriptions and definitions of epitopes, dependenton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described at the amino acid level, e.g., determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues. Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue is shared by the epitopes.

Epitopes characterized by competition binding are said to be overlappingif the binding of the corresponding antibodies are mutually exclusive,i.e., binding of one antibody excludes simultaneous or consecutivebinding of the other antibody. The epitopes are said to be separate(unique) if the antigen is able to accommodate binding of bothcorresponding antibodies simultaneously.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the antibody whichspecifically binds an antigen, i.e., the amino acid residues on theantibody which make contact with the antigen (IL-2) as “contact” isdefined elsewhere herein.

The epitope and paratope for a given antibody/antigen pair may beidentified by routine methods. For example, the general location of anepitope may be determined by assessing the ability of an antibody tobind to different fragments or variant IL-2 polypeptides. The specificamino acids within IL-2 that make contact with an antibody (epitope) andthe specific amino acids in an antibody that make contact with IL-2(paratope) may also be determined using routine methods, such as thosedescribed in the examples. For example, the antibody and target moleculemay be combined and the antibody/antigen complex may be crystallized.The crystal structure of the complex may be determined and used toidentify specific sites of interaction between the antibody and itstarget.

An antibody according to the current disclosure may bind to the sameepitope or domain of IL-2 as the antibodies of the disclosure that arespecifically disclosed herein. For example, other yet unidentifiedantibodies of the disclosure may be identified by comparing theirbinding to IL-2 with that of any of the following monoclonal antibodies:F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01, F5.1.11.02,F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07, F5.1.11.08,F5.1.11.09, F5.1.9.5, or d1C7, and variants thereof; or by comparing thefunction of yet unidentified antibodies with that of the antibodiesdescribed herein; and/or by comparing the epitope/contact residues onIL-2 of yet unidentified antibodies with those of the antibodies of thedisclosure. Analyses and assays that may be used for the purpose of suchidentification include assays assessing the competition for binding ofIL-2 between the antibody of interest and IL-2 receptor, in biologicalactivity assays as described in Examples 1-5, and in analysis of thecrystal structure of the antibody.

An antibody of the disclosure may have the ability to compete orcross-compete with another antibody of the disclosure for binding toIL-2 as described herein. For example, an antibody of the disclosure maycompete or cross-compete with antibodies described herein for binding toIL-2, or to a suitable fragment or variant of IL-2 that is bound by theantibodies disclosed herein.

That is, if a first antibody competes with a second antibody for bindingto IL-2, but it does not compete where the second antibody is firstbound to IL-2, it is deemed to “compete” with the second antibody (alsoreferred to as unidirectional competition). Where an antibody competeswith another antibody regardless of which antibody is first bound toIL-2, then the antibody “cross-competes” for binding to IL-2 with theother antibody. Such competing or cross-competing antibodies can beidentified based on their ability to compete/cross-compete with a knownantibody of the disclosure in standard binding assays. For example, SPRe.g. by using a Biacore™ system, ELISA assays or flow cytometry may beused to demonstrate competition/cross-competition. Suchcompetition/cross-competition may suggest that the two antibodies bindto identical, overlapping or similar epitopes.

An antibody of the disclosure may therefore be identified by a methodthat comprises a binding assay which assesses whether or not a testantibody is able to compete/cross-compete with a reference antibody ofthe disclosure (e.g., F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062,F5.11.1.01, F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06,F5.1.11.07, F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7) for a bindingsite on the target molecule. Methods for carrying out competitivebinding assays are disclosed herein and/or are well known in the art.For example they may involve binding a reference antibody of thedisclosure to a target molecule using conditions under which theantibody can bind to the target molecule. The antibody/target complexmay then be exposed to a test/second antibody and the extent to whichthe test antibody is able to displace the reference antibody of thedisclosure from antibody/target complexes may be assessed. Analternative method may involve contacting a test antibody with a targetmolecule under conditions that allow for antibody binding, then adding areference antibody of the disclosure that is capable of binding thattarget molecule and assessing the extent to which the reference antibodyof the disclosure is able to displace the test antibody fromantibody/target complexes or to simultaneously bind to the target (i.e.,non-competing antibody).

The ability of a test antibody to inhibit the binding of a referenceantibody of the disclosure to the target demonstrates that the testantibody can compete with a reference antibody of the disclosure forbinding to the target and thus that the test antibody binds to the same,or substantially the same, epitope or region on the IL-2 protein as thereference antibody of the disclosure. A test antibody that is identifiedas competing with a reference antibody of the disclosure in such amethod is also an antibody of the present disclosure. The fact that thetest antibody can bind IL-2 in the same region as a reference antibodyof the disclosure and can compete with the reference antibody of thedisclosure suggests that the test antibody may act as a ligand at thesame binding site as the antibody of the disclosure and that the testantibody may therefore mimic the action of the reference antibody andis, thus, an antibody of the disclosure. This can be confirmed bycomparing the activity of IL-2 in the presence of the test antibody withthe activity of IL-2 in the presence of the reference antibody underotherwise identical conditions, using an assay as more fully describedelsewhere herein.

The reference antibody of the disclosure may be an antibody as describedherein, such as F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01,F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07,F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7, or any variant, or portionthereof, as described herein that retains the ability to bind to IL-2.An antibody of the disclosure may bind to the same epitope as thereference antibodies described herein or any variant or portion thereofas described herein that retains the ability to bind to IL-2.

As stated previously elsewhere herein, specific binding may be assessedwith reference to binding of the antibody to a molecule that is not thetarget. This comparison may be made by comparing the ability of anantibody to bind to the target and to another molecule. This comparisonmay be made as described above in an assessment of K_(D) or K_(i). Theother molecule used in such a comparison may be any molecule that is notthe target molecule. In some embodiments, the other molecule is notidentical to the target molecule. In some embodiments, the targetmolecule is not a fragment of the target molecule.

The other molecule used to determine specific binding may be unrelatedin structure or function to the target. For example, the other moleculemay be an unrelated material or accompanying material in theenvironment.

The other molecule used to determine specific binding may be anothermolecule involved in the same in vivo pathway as the target molecule,i.e., IL-2. By ensuring that the antibody of the disclosure hasspecificity for IL-2 over another such molecule, unwanted in vivocross-reactivity may be avoided.

The antibody of the disclosure may retain the ability to bind to somemolecules that are related to the target molecule.

Alternatively, the antibody of the disclosure may have specificity for aparticular target molecule. For example, it may bind to one targetmolecule as described herein, but may not bind, or may bind withsignificantly reduced affinity to a different target molecule asdescribed herein. For example, a full length mature human IL-2 may beused as the target, but the antibody that binds to that target may beunable to bind to or may bind with lesser affinity to, e.g. other IL-2proteins from other species, such as other mammalian IL-2. In someembodiments, the antibody binds to both human and mouse IL-2.

Polypeptide or antibody “fragments” or “portions” according to thedisclosure may be made by truncation, e.g., by removal of one or moreamino acids from the N and/or C-terminal ends of a polypeptide. Up to10, up to 20, up to 30, up to 40 or more amino acids may be removed fromthe N and/or C terminal in this way. Portions may also be generated byone or more internal deletions.

An antibody of the disclosure may be, or may comprise, a portion of, anyone of antibodies F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01,F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07,F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7, or a variant thereof. Theantibody of the disclosure may be or may comprise an antigen-bindingportion of this antibody or a variant thereof. For example, the antibodyof the disclosure may be a Fab portion of this antibody or a variantthereof or may be a single chain antibody derived from this antibody ora variant thereof.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to30 or more amino acid substitutions and/or deletions and/or insertionsfrom the specific sequences and portions discussed above. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Insertion” variants may comprisethe insertion of individual amino acids, insertion of small groups ofamino acids such as 2, 3, 4 or 5 amino acids, or insertion of largeramino acid regions, such as the insertion of specific amino acid domainsor other features. “Substitution” variants in some embodiments involvethe replacement of one or more amino acids with the same number of aminoacids and making conservative amino acid substitutions. For example, anamino acid may be substituted with an alternative amino acid havingsimilar properties, for example, another basic amino acid, anotheracidic amino acid, another neutral amino acid, another charged aminoacid, another hydrophilic amino acid, another hydrophobic amino acid,another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as described below.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable domains, but framework alterations are alsocontemplated. Conservative substitutions are shown in Table 1 under theheading of “conservative substitutions.” If such substitutions result ina change in biological activity, then more substantial changes,denominated “exemplary substitutions” shown below, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 1 Amino Acid Substitutions Conservative Original ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Noneucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a P-sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gin;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

One type of substitution, for example, that may be made is to change oneor more cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. Forexample, there can be a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant region of an antibody. In some embodiments,the cysteine is canonical. Any cysteine residue not involved inmaintaining the proper conformation of the antibody also may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant cross-linking. Conversely, cysteinebond(s) may be added to the antibody to improve its stability,particularly where the antibody is an antibody portion such as an Fvportion.

The disclosure also provides methods of generating, selecting, andmaking IL-2 antibodies. The antibodies of this disclosure can be made byprocedures known in the art. In some embodiments, antibodies may be maderecombinantly and expressed using any method known in the art. Thedisclosure also provides methods of generating, selecting, and makinghIL-2 antibodies. The antibodies of this disclosure can be made byprocedures known in the art. In some embodiments, antibodies may be maderecombinantly and expressed using any method known in the art.

In some embodiments, antibodies may be prepared and selected by phagedisplay technology. See, for example, U.S. Pat. Nos. 5,565,332;5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev.Immunol. 12:433-455, 1994. Alternatively, the phage display technology(McCafferty et al., Nature 348:552-553, 1990) can be used to producehuman antibodies and antibody portions in vitro, from immunoglobulinvariable (V) domain gene repertoires from unimmunized donors. Accordingto this technique, antibody V domain genes are cloned in-frame intoeither a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyportions on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B cell.Phage display can be performed in a variety of formats; for review see,e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571, 1993. Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature 352:624-628,1991, isolated a diverse array of anti-oxazolone antibodies from a smallrandom combinatorial library of V genes derived from the spleens ofimmunized mice. A repertoire of V genes from human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Mark et al., 1991, J. Mol. Biol. 222:581-597, or Griffithet al., 1993, EMBO J. 12:725-734. In a natural immune response, antibodygenes accumulate mutations at a high rate (somatic hypermutation). Someof the changes introduced will confer higher affinity, and B cellsdisplaying high-affinity surface immunoglobulin are preferentiallyreplicated and differentiated during subsequent antigen challenge. Thisnatural process can be mimicked by employing the technique known as“chain shuffling.” (Marks et al., 1992, Bio/Technol. 10:779-783). Inthis method, the affinity of “primary” human antibodies obtained byphage display can be improved by sequentially replacing the heavy andlight chain V region genes with repertoires of naturally occurringvariants (repertoires) of V domain genes obtained from unimmunizeddonors. This technique allows the production of antibodies and antibodyportions with affinities in the pM-nM range. A strategy for making verylarge phage antibody repertoires (also known as “the mother-of-alllibraries”) has been described by Waterhouse et al., Nucl. Acids Res.21:2265-2266, 1993. Gene shuffling can also be used to derive humanantibodies from rodent antibodies, where the human antibody has similaraffinities and specificities to the starting rodent antibody. Accordingto this method, which is also referred to as “epitope imprinting”, theheavy or light chain V domain gene of rodent antibodies obtained byphage display technique is replaced with a repertoire of human V domaingenes, creating rodent-human chimeras. Selection on antigen results inisolation of human variable domains capable of restoring a functionalantigen-binding site, i.e., the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT Publication No.WO 93/06213). Unlike traditional humanization of rodent antibodies byCDR grafting, this technique provides completely human antibodies, whichhave no framework or CDR residues of rodent origin.

In some embodiments, antibodies may be made using hybridoma technology.It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. The route and schedule of immunization of the host animal aregenerally in keeping with established and conventional techniques forantibody stimulation and production, as further described herein.Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., 1975, Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the IL-2 monoclonal antibodies of the subjectdisclosure. The hybridomas or other immortalized B-cells are expandedand subcloned, if desired, and supernatants are assayed foranti-immunogen activity by conventional immunoassay procedures (e.g.,radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for IL-2, or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with an IL-2 polypeptide, or aportion containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, the IL-2 antibody (monoclonal or polyclonal) of interest maybe sequenced and the polynucleotide sequence may then be cloned into avector for expression or propagation. The sequence encoding the antibodyof interest may be maintained in vector in a host cell and the host cellcan then be expanded and frozen for future use. Production ofrecombinant monoclonal antibodies in cell culture can be carried outthrough cloning of antibody genes from B cells by means known in theart. See, e.g. Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S.Pat. No. 7,314,622.

The plasmids indicated in Table 2 are being deposited under terms inaccordance with the Budapest Treaty with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. Theplasmids have been assigned the following accession numbers:

TABLE 2 Strain Antibody Designation Deposit Date F5.1.11.02 VHF5.1.11.02-VH Sep. 8, 2016 F5.1.11.02 VL F5.1.11.02-VL Sep. 8, 2016

In some embodiments, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. Antibodies may also be customizedfor use, for example, in dogs, cats, primate, equines and bovines.

In some embodiments, fully human antibodies may be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods231:147, 1999. Methods for making derivatives of antibodies, e.g.,domain, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for IL-2.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). In some embodiments, thehybridoma cells serve as a source of such DNA. Once isolated, the DNAmay be placed into expression vectors (such as expression vectorsdisclosed in PCT Publication No. WO 87/04462), which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. See, e.g., PCTPublication No. WO 87/04462. The DNA also may be modified, for example,by substituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences, Morrisonet al., Proc. Nat. Acad. Sci. 81:6851, 1984, or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. In that manner, “chimeric” or“hybrid” antibodies are prepared that have the binding specificity of a,IL-2 antibody herein.

Antibody portions can be produced by proteolytic or other degradation ofthe antibodies, by recombinant methods (i.e., single or fusionpolypeptides) as described above or by chemical synthesis. Polypeptidesof the antibodies, especially shorter polypeptides up to about 50 aminoacids, are conveniently made by chemical synthesis. Methods of chemicalsynthesis are known in the art and are commercially available. Forexample, an antibody could be produced by an automated polypeptidesynthesizer employing the solid phase method. See also, U.S. Pat. Nos.5,807,715; 4,816,567; and 6,331,415.

In some embodiments, a polynucleotide comprises a sequence encoding theheavy chain and/or the light chain variable domains of IL-2 antibody ofthe present disclosure. The sequence encoding the antibody of interestmay be maintained in a vector in a host cell and the host cell can thenbe expanded and frozen for future use. Vectors (including expressionvectors) and host cells are further described herein.

The disclosure includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al.,1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene,169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson etal., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol.Biol., 226:889-896; and PCT Publication No. WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody and/or altering (such as improving) the binding affinityof a polypeptide, such as an antibody, termed “library scanningmutagenesis”. Generally, library scanning mutagenesis works as follows.One or more amino acid positions in the CDR are replaced with two ormore (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) amino acids using art recognized methods. This generatessmall libraries of clones (in some embodiments, one for every amino acidposition that is analyzed), each with a complexity of two or moremembers (if two or more amino acids are substituted at every position).Generally, the library also includes a clone comprising the native(unsubstituted) amino acid. A small number of clones, e.g., about 20-80clones (depending on the complexity of the library), from each libraryare screened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased, orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using, forexample, Biacore™ surface plasmon resonance analysis, which detectsdifferences in binding affinity of about 2-fold or greater, Kinexa®Biosensor, scintillation proximity assays, ELISA, ORIGEN® immunoassay,fluorescence quenching, fluorescence transfer, and/or yeast display.Binding affinity may also be screened using a suitable bioassay.Biacore™ is particularly useful when the starting antibody already bindswith a relatively high affinity, for example a K_(D) of about 10 nM orlower.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position). Insome embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids exceptcysteine using art recognized mutagenesis methods

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., 1993, Gene137(1):109-18.

The CDR may be heavy chain variable domain (VH) CDR3 and/or light chainvariable domain (VL) CDR3. The CDR may be one or more of VHCDR1, VHCDR2,VH CDR3, VLCDR1, VLCDR2, and/or VLCDR3. The CDR may be a Kabat CDR, aChothia CDR, an extended CDR, an AbM CDR, a contact CDR, or aconformational CDR.

In some embodiments, the library is made according to SEQ ID NOs: 71 and72.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acids (including all 20amino acids), and positions in the CDRs which cannot be changed or whichcan only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid at thatposition, and may further include additional substitutions at thatposition, depending on the complexity of the library that is desired, orpermitted using the desired screening or selection method.

In addition, if desired, adjacent amino acid position can be randomizedto at least two or more amino acids. Randomization of adjacent aminoacids may permit additional conformational flexibility in the mutantCDR, which may in turn, permit or facilitate the introduction of alarger number of improving mutations. The library may also comprisesubstitution at positions that did not show improved affinity in thefirst round of screening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using Biacore, Kinexa™ biosensor analysis, andselection using any method known in the art for selection, includingphage display, yeast display, and ribosome display.

To express the IL-2 antibodies of the present disclosure, DNA fragmentsencoding VH and VL regions can first be obtained using any of themethods described above. Various modifications, e.g. mutations,deletions, and/or additions can also be introduced into the DNAsequences using standard methods known to those of skill in the art. Forexample, mutagenesis can be carried out using standard methods, such asPCR-mediated mutagenesis, in which the mutated nucleotides areincorporated into the PCR primers such that the PCR product contains thedesired mutations or site-directed mutagenesis.

The disclosure encompasses modifications to the variable domains and theCDRs indicated in Table 7. For example, the disclosure includesantibodies comprising functionally equivalent variable domains and CDRswhich do not significantly affect their properties as well as variantswhich have enhanced or decreased activity and/or affinity. For example,the amino acid sequence may be mutated to obtain an antibody with thedesired binding affinity to IL-2. In some embodiments, the amino acidsequence may be mutated to obtain an antibody with the desired bindingaffinity to IL-2. Examples of modified polypeptides include polypeptideswith conservative substitutions of amino acid residues, one or moredeletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

The antibodies may also be modified, e.g., in the variable domains ofthe heavy and/or light chains, e.g., to alter a binding property of theantibody. Changes in the variable domain can alter binding affinityand/or specificity. In some embodiments, no more than one to fiveconservative amino acid substitutions are made within a CDR domain. Inother embodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. For example, a mutation maybe made in one or more of the CDR regions to increase or decrease theK_(D) of the antibody for IL-2, to increase or decrease k_(off), or toalter the binding specificity of the antibody. Techniques insite-directed mutagenesis are well-known in the art. See, e.g., Sambrooket al. and Ausubel et al., supra.

A modification or mutation may also be made in a framework region orconstant region to increase the half-life of an IL-2 antibody. See,e.g., PCT Publication No. WO 00/09560. A mutation in a framework regionor constant region can also be made to alter the immunogenicity of theantibody, to provide a site for covalent or non-covalent binding toanother molecule, or to alter such properties as complement fixation,FcR binding and antibody-dependent cell-mediated cytotoxicity. Accordingto the disclosure, a single antibody may have mutations in any one ormore of the CDRs or framework regions of the variable domain or in theconstant region.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular,antibodies produced by CHO cells with tetracycline-regulated expressionof β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GIcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, NatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example, usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

In some embodiments, the antibody comprises a modified constant regionthat has increased or decreased binding affinity to a human Fc gammareceptor, is immunologically inert or partially inert, e.g., does nottrigger complement mediated lysis, does not stimulate antibody-dependentcell mediated cytotoxicity (ADCC), or does not activate microglia; orhas reduced activities (compared to the unmodified antibody) in any oneor more of the following: triggering complement mediated lysis,stimulating ADCC, or activating microglia. Different modifications ofthe constant region may be used to achieve optimal level and/orcombination of effector functions. See, for example, Morgan et al.,Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000;Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al.,Immunological Reviews 163:59-76, 1998. In some embodiments, the constantregion is modified as described in Eur. J. Immunol., 1999, 29:2613-2624;PCT Application No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8.

In some embodiments, an antibody constant region can be modified toavoid interaction with Fc gamma receptor and the complement and immunesystems. The techniques for preparation of such antibodies are describedin WO 99/58572. For example, the constant region may be engineered tomore resemble human constant regions to avoid immune response if theantibody is used in clinical trials and treatments in humans. See, e.g.,U.S. Pat. Nos. 5,997,867 and 5,866,692.

In some embodiments, the constant region is modified as described inEur. J. Immunol., 1999, 29:2613-2624; PCT Application No.PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In suchembodiments, the Fc can be human IgG₂ or human IgG₄. The Fc can be humanIgG₂ containing the mutation A330P331 to S330S331 (IgG_(2Δa)), in whichthe amino acid residues are numbered with reference to the wild typeIgG₂ sequence. Eur. J. Immunol., 1999, 29:2613-2624. In someembodiments, the antibody comprises a constant region of IgG₄ comprisingthe following mutations (Armour et al., 2003, Molecular Immunology 40585-593): E233F234L235 to P233V234A235 (IgG_(4Δc)), in which thenumbering is with reference to wild type IgG₄. In yet anotherembodiment, the Fc is human IgG₄ E233F234L235 to P233V234A235 withdeletion G236 (IgG_(4Δb)). In another embodiment, the Fc is any humanIgG₄ Fc (IgG₄, IgG_(4Δb) or IgG_(4Δc)) containing hinge stabilizingmutation S228 to P228 (Aalberse et al., 2002, Immunology 105, 9-19).

In some embodiments, the antibody comprises a human heavy chain IgG₂constant region comprising the following mutations: A330P331 to S330S331(amino acid numbering with reference to the wild type IgG₂ sequence).Eur. J. Immunol., 1999, 29:2613-2624. In still other embodiments, theconstant region is aglycosylated for N-linked glycosylation. In someembodiments, the constant region is aglycosylated for N-linkedglycosylation by mutating the oligosaccharide attachment residue and/orflanking residues that are part of the N-glycosylation recognitionsequence in the constant region. For example, N-glycosylation site N297may be mutated to, e.g., A, Q, K, or H. See, Tao et al., J. Immunology143: 2595-2601, 1989; and Jefferis et al., Immunological Reviews163:59-76, 1998. In some embodiments, the constant region isaglycosylated for N-linked glycosylation. The constant region may beaglycosylated for N-linked glycosylation enzymatically (such as removingcarbohydrate by enzyme PNGase), or by expression in a glycosylationdeficient host cell.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572. These antibodiescomprise, in addition to a binding domain directed at the targetmolecule, an effector domain having an amino acid sequence substantiallyhomologous to all or part of a constant region of a human immunoglobulinheavy chain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. In some embodiments, theeffector domain is capable of specifically binding FcRn and/or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain CH2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy.

The disclosure also provides an antibody constant domain that may befurther modified. It is known that variants of the Fc region, e.g.,amino acid substitutions, insertions, and/or additions and/or deletions,enhance or diminish effector function. See, e.g., Presta et al, 2002,Biochem. Soc. Trans. 30:487-490; Strohl, 2009, Curr. Opin. Biotechnol.20(6):685-691; U.S. Pat. Nos. 5,624,821, 5,648,260, 5,885,573,6,737,056, 7,317,091; PCT publication Nos. WO 99/58572, WO 00/42072, WO04/029207, WO 2006/105338, WO 2008/022152, WO 2008/150494, WO2010/033736; U.S. Patent Application Publication Nos. 2004/0132101,2006/0024298, 2006/0121032, 2006/0235208, 2007/0148170; Armour et al.,1999, Eur. J. Immunol. 29(8):2613-2624 (reduced ADCC and CDC); Shieldset al., 2001, J. Biol. Chem. 276(9):6591-6604 (reduced ADCC and CDC);Idusogie et al., 2000, J. Immunol. 164(8):4178-4184 (increased ADCC andCDC); Steurer et al., 1995, J. Immunol. 155(3):1165-1174 (reduced ADCCand CDC); Idusogie et al., 2001, J. Immunol. 166(4):2571-2575 (increasedADCC and CDC); Lazar et al., 2006, Proc. Natl. Acad. Sci. USA 103(11):4005-4010 (increased ADCC); Ryan et al., 2007, Mol. Cancer. Ther., 6:3009-3018 (increased ADCC); Richards et al., 2008, Mol. Cancer Ther.7(8):2517-2527.

In some embodiments, the antibody comprises a modified constant regionthat has increased binding affinity for FcRn and/or an increased serumhalf-life as compared with the unmodified antibody.

In a process known as “germlining”, certain amino acids in the VH and VLsequences can be mutated to match those found naturally in germline VHand VL sequences. In particular, the amino acid sequences of theframework regions in the VH and VL sequences can be mutated to match thegermline sequences to reduce the risk of immunogenicity when theantibody is administered. Germline DNA sequences for human VH and VLgenes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994, Eur. J.Immunol. 24:827-836).

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant regionof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues. Inanother example, the C-terminal lysine of the heavy chain of an IL-2antibody of the disclosure can be cleaved or otherwise removed. Invarious embodiments of the disclosure, the heavy and light chains of theantibodies may optionally include a signal sequence.

Once DNA fragments encoding the VH and VL segments of the presentdisclosure are obtained, these DNA fragments can be further manipulatedby standard recombinant DNA techniques, for example to convert thevariable domain genes to full-length antibody chain genes, to Fabfragment genes, or to a scFv gene. In these manipulations, a VL- orVH-encoding DNA fragment is operatively linked to another DNA fragmentencoding another protein, such as an antibody constant region or aflexible linker. The term “operatively linked”, as used in this context,is intended to mean that the two DNA fragments are joined such that theamino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG₁, IgG₂,IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region, but in someembodiments, is an IgG₁ or IgG₂ constant region. The IgG constant regionsequence can be any of the various alleles or allotypes known to occuramong different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17).These allotypes represent naturally occurring amino acid substitution inthe IgG1 constant regions. For a Fab fragment heavy chain gene, theVH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region. The CH1 heavy chainconstant region may be derived from any of the heavy chain genes.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region. The kappa constant region maybe any of the various alleles known to occur among differentindividuals, such as Inv(1), Inv(2), and Inv(3). The lambda constantregion may be derived from any of the three lambda genes.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker suchthat the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (See e.g., Bird et al., 1988, Science 242:423-426; Huston et al.,1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990,Nature 348:552-554. Linkers of other sequences have been designed andused (Bird et al., 1988, supra). Linkers can in turn be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. The single chain antibody may be monovalent, if only a singleVH and VL are used, bivalent, if two VH and VL are used, or polyvalent,if more than two VH and VL are used. Bispecific or polyvalent antibodiesmay be generated that bind specifically to IL-2 and to another molecule.The single chain variants can be produced either recombinantly orsynthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

Other forms of single chain antibodies, such as diabodies, are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al., 1993, Proc. Natl. Acad Sci. USA90:6444-6448; and Poljak, R. J., et al., 1994, Structure 2:1121-1123).

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the disclosure. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT Publication Nos. WO91/00360 and WO 92/200373; and EP 03089). Heteroconjugate antibodies maybe made using any convenient cross-linking methods. Suitablecross-linking agents and techniques are well known in the art, and aredescribed in U.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

The disclosure also encompasses fusion proteins comprising one or moreportions or regions from the antibodies disclosed herein. In someembodiments, a fusion antibody may be made that comprises all or aportion of an IL-2 antibody of the disclosure linked to anotherpolypeptide. In another embodiment, only the variable domains of theIL-2 antibody are linked to the polypeptide. In another embodiment, theVH domain of an IL-2 antibody is linked to a first polypeptide, whilethe VL domain of an IL-2 antibody is linked to a second polypeptide thatassociates with the first polypeptide in a manner such that the VH andVL domains can interact with one another to form an antigen bindingsite. In another embodiment, the VH domain is separated from the VLdomain by a linker such that the VH and VL domains can interact with oneanother. The VH-linker-VL antibody is then linked to the polypeptide ofinterest. In addition, fusion antibodies can be created in which two (ormore) single-chain antibodies are linked to one another. This is usefulif one wants to create a divalent or polyvalent antibody on a singlepolypeptide chain, or if one wants to create a bispecific antibody.

In some embodiments, a fusion polypeptide is provided that comprises atleast contiguous amino acids of the variable light chain region shown inSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,or 72 and/or at least 10 amino acids of the variable heavy chain regionshown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, or 71. In other embodiments, a fusion polypeptide is providedthat comprises at least about 10, at least about 15, at least about 20,at least about 25, or at least about 30 contiguous amino acids of thevariable light chain region and/or at least about 10, at least about 15,at least about 20, at least about 25, or at least about contiguous aminoacids of the variable heavy chain region. In another embodiment, thefusion polypeptide comprises one or more CDR(s). In still otherembodiments, the fusion polypeptide comprises VH CDR3 and/or VL CDR3.For purposes of this disclosure, a fusion protein contains one or moreantibodies and another amino acid sequence to which it is not attachedin the native molecule, for example, a heterologous sequence or ahomologous sequence from another region. Exemplary heterologoussequences include, but are not limited to a “tag” such as a FLAG tag ora 6His tag (SEQ ID NO: 223). Tags are well known in the art.

A fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the fusion proteinsof this disclosure are made by preparing and expressing a polynucleotideencoding them using recombinant methods described herein, although theymay also be prepared by other means known in the art, including, forexample, chemical synthesis.

In other embodiments, other modified antibodies may be prepared usingnucleic acid molecules encoding an IL-2 antibody. For instance, “Kappabodies” (111 et al., 1997, Protein Eng. 10:949-57), “Minibodies” (Martinet al., 1994, EMBO J. 13:5303-9), “Diabodies” (Holliger et al., supra),or “Janusins” (Traunecker et al., 1991, EMBO J. 10:3655-3659 andTraunecker et al., 1992, Int. J. Cancer (Suppl.) 7:51-52) may beprepared using standard molecular biological techniques following theteachings of the specification.

For example, bispecific antibodies, monoclonal antibodies that havebinding specificities for at least two different antigens, can beprepared using the antibodies disclosed herein. Methods for makingbispecific antibodies are known in the art (see, e.g., Suresh et al.,1986, Methods in Enzymology 121:210). For example, bispecific antibodiesor antigen-binding portions can be produced by fusion of hybridomas orlinking of Fab′ portions. See, e.g., Songsivilai & Lachmann, 1990, Clin.Exp. Immunol. 79:315-321, and Kostelny et al., 1992, J. Immunol.148:1547-1553. Traditionally, the recombinant production of bispecificantibodies was based on the coexpression of two immunoglobulin heavychain-light chain pairs, with the two heavy chains having differentspecificities (Millstein and Cuello, 1983, Nature 305, 537-539). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of IL-2. In some embodiments, the modified antibodiesdescribed above are prepared using one or more of the variable domainsor CDR regions from an IL-2 antibody provided herein.

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantregion sequences. In some embodiments, the fusion is with animmunoglobulin heavy chain constant region, comprising at least part ofthe hinge, CH2 and CH3 regions. In some embodiments, the first heavychain constant region (CH1), containing the site necessary for lightchain binding, is present in at least one of the fusions. DNAs encodingthe immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are cotransfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide portions in embodiments when unequal ratios of thethree polypeptide chains used in the construction provide the optimumyields. It is, however, possible to insert the coding sequences for twoor all three polypeptide chains in one expression vector when theexpression of at least two polypeptide chains in equal ratios results inhigh yields or when the ratios are of no particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690.

This disclosure also provides compositions comprising antibodiesconjugated (for example, linked) to an agent that facilitate coupling toa solid support (such as biotin or avidin). For simplicity, referencewill be made generally to antibodies with the understanding that thesemethods apply to any of the IL-2 binding embodiments described herein.Conjugation generally refers to linking these components as describedherein. The linking (which is generally fixing these components inproximate association at least for administration) can be achieved inany number of ways. For example, a direct reaction between an agent andan antibody is possible when each possesses a substituent capable ofreacting with the other. For example, a nucleophilic group, such as anamino or sulfhydryl group, on one may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide) onthe other.

The antibodies can be bound to many different carriers. Carriers can beactive and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the disclosure. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

An antibody or polypeptide of this disclosure may be linked to alabeling agent such as a fluorescent molecule, a radioactive molecule orany others labels known in the art. Labels are known in the art whichgenerally provide (either directly or indirectly) a signal.

The amino acid sequences of the light chain variable domain (VL) andheavy chain variable domains (VH) of the IL-2 antibodies disclosedherein are summarized in Table 7 by sequence identification number.

An antibody of the disclosure may comprise both:

a) a VH comprising the amino acid sequence of SEQ ID NO:1, and a VLcomprising the amino acid sequence of SEQ ID NO:2,b) a VH comprising the amino acid sequence of SEQ ID NO:3, and a VLcomprising the amino acid sequence of SEQ ID NO:4,c) a VH comprising the amino acid sequence of SEQ ID NO:5, and a VLcomprising the amino acid sequence of SEQ ID NO:6,d) a VH comprising the amino acid sequence of SEQ ID NO:7, and a VLcomprising the amino acid sequence of SEQ ID NO:8,e) a VH comprising the amino acid sequence of SEQ ID NO:9, and a VLcomprising the amino acid sequence of SEQ ID NO:10,f) a VH comprising the amino acid sequence of SEQ ID NO:11, and a VLcomprising the amino acid sequence of SEQ ID NO:12,g) a VH comprising the amino acid sequence of SEQ ID NO:13, and a VLcomprising the amino acid sequence of SEQ ID NO:14,h) a VH comprising the amino acid sequence of SEQ ID NO:15, and a VLcomprising the amino acid sequence of SEQ ID NO:16,i) a VH comprising the amino acid sequence of SEQ ID NO:17, and a VLcomprising the amino acid sequence of SEQ ID NO:18,j) a VH comprising the amino acid sequence of SEQ ID NO:19, and a VLcomprising the amino acid sequence of SEQ ID NO:20,k) a VH comprising the amino acid sequence of SEQ ID NO:21, and a VLcomprising the amino acid sequence of SEQ ID NO:22,l) a VH comprising the amino acid sequence of SEQ ID NO:23, and a VLcomprising the amino acid sequence of SEQ ID NO:24,m) a VH comprising the amino acid sequence of SEQ ID NO:25, and a VLcomprising the amino acid sequence of SEQ ID NO:26,n) a VH comprising the amino acid sequence of SEQ ID NO:27, and a VLcomprising the amino acid sequence of SEQ ID NO:28,o) a VH comprising the amino acid sequence of SEQ ID NO:29, and a VLcomprising the amino acid sequence of SEQ ID NO:30,p) a VH comprising the amino acid sequence of SEQ ID NO:31, and a VLcomprising the amino acid sequence of SEQ ID NO:32 orq) a VH comprising the amino acid sequence of SEQ ID NO:71, and a VLcomprising the amino acid sequence of SEQ ID NO:72.

In another aspect, the antibody comprises a variant of these sequences,wherein such variants can include both conservative and non-conservativesubstitutions, deletions, and/or additions, and typically includepeptides that share at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 87%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to any of the specific sequences disclosed herein.

For example, in one aspect, the disclosure provides an isolated antibodyor antigen-binding portion thereof that comprises a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72, or a variantthereof. In one aspect, said antibody variant comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservativesubstitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 additions and/or deletions to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72. In a further aspect,said variant shares at least 65%, at least 75%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72, and wherein saidantibody or antigen-binding portion specifically binds IL-2. In someembodiments, said antibody or antigen-binding portion specifically bindshIL-2.

In a further aspect, the disclosure provides an isolated antibody orantigen-binding portion thereof that comprises a V_(H) chain amino acidsequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71, or a variantthereof. In one aspect, said antibody variant comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservativesubstitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 additions and/or deletions to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71. In a further aspect,said variant shares at least 65%, at least 75%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71, and wherein saidantibody or antigen-binding portion specifically binds IL-2. In someembodiments, said antibody or antigen-binding portion specifically bindshIL-2.

An antibody of the disclosure may comprise a heavy chain comprising a VHcomprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71,wherein the antibody further comprises a heavy chain constant domain. Asmore fully set forth elsewhere herein, the antibody heavy chain constantdomain can be selected from an IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgE, IgM orIgD constant region, but in some embodiments, is an IgG₁ or IgG₂constant region. The IgG constant region sequence can be any of thevarious alleles or allotypes known to occur among different individuals,such as Gm(1), Gm(2), Gm(3), and Gm(17). For a Fab portion heavy chaingene, the VH-encoding DNA can be operatively linked to another DNAmolecule encoding only the heavy chain CH1 constant region. The CH1heavy chain constant region may be derived from any of the heavy chaingenes.

In one aspect, the antibody may comprise a heavy chain comprising a VHselected from a VH comprising the amino acid sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ IDNO:71, and further comprising the IgG1 constant domain comprising atriple mutation decreasing or abolishing Fc effector function (hIgG1-3m;SEQ ID NO:2). In one aspect, said antibody variant comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative ornon-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 additions and/or deletions to the full lengthheavy chain. In a further aspect, said variant shares at least 65%, atleast 75%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity with the fulllength heavy chain, and wherein said antibody or antigen-binding portionspecifically binds IL-2.

An antibody of the disclosure may comprise a light chain comprising a VLcomprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72,wherein the antibody further comprises a light chain constant domain. Asmore fully set forth elsewhere herein, the antibody light chain constantdomain can be selected from a CK or CA constant region, for example theCA constant region of SEQ ID NO:1. In one aspect, said antibody variantcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15conservative or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions to thefull length light chain. In a further aspect, said variant shares atleast 65%, at least 75%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identitywith the full length light chain, and wherein said antibody orantigen-binding portion specifically binds IL-2.

An antibody of the disclosure may comprise a portion of one of the VL orVH amino acid sequences shown in Table 7. For example, an antibody ofthe disclosure may comprise a portion of at least 7, at least 8, atleast 9, at least 10, at least 12, at least 15, at least 18, at least 20or at least 25 consecutive amino acids from a VH comprising SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ IDNO:71, or from a VL comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72. Such a portion willpreferably retain one or more of the functions discussed above, such asthe ability to bind to IL-2. In some embodiments, such a portion willpreferably retain one or more of the functions discussed above, such asthe ability to bind to hIL-2.

In some embodiments, the antibody of the disclosure comprises the VHCDR1, CDR2, and CDR3 in accordance with Kabat, Chothia, or extendedsequences and/or VL CDR1, CDR2, and CDR3 in accordance with Kabat and/orChothia amino acid sequences as shown in Table 7. In some embodiments,the antibody of the disclosure comprises a VH CDR1 in accordance withthe following Kabat amino acid sequences: SEQ ID NO:73, SEQ ID NO:82,SEQ ID NO:91, SEQ ID NO:100, SEQ ID NO:109, SEQ ID NO:118, SEQ IDNO:127, SEQ ID NO:136, SEQ ID NO:145, SEQ ID NO:154, SEQ ID NO:163, SEQID NO:172, SEQ ID NO:181, SEQ ID NO:190, SEQ ID NO:199, or SEQ IDNO:208. In some embodiments, the antibody of the disclosure comprises aVH CDR1 in accordance with the following Chothia amino acid sequences:SEQ ID NO:74, SEQ ID NO:83, SEQ ID NO:92, SEQ ID NO:101, SEQ ID NO:110,SEQ ID NO:119, SEQ ID NO:128, SEQ ID NO:137, SEQ ID NO:146, SEQ IDNO:155, SEQ ID NO:164, SEQ ID NO:173, SEQ ID NO:182, SEQ ID NO:191, SEQID NO:200, or SEQ ID NO:209. In some embodiments, the antibody of thedisclosure comprises a VH CDR1 in accordance with the followingcombination amino acid sequences: SEQ ID NO:75, SEQ ID NO:84, SEQ IDNO:93, SEQ ID NO:102, SEQ ID NO:111, SEQ ID NO:120, SEQ ID NO:129, SEQID NO:138, SEQ ID NO:147, SEQ ID NO:156, SEQ ID NO:165, SEQ ID NO:174,SEQ ID NO:183, SEQ ID NO:192, SEQ ID NO:201, or SEQ ID NO:210. In someembodiments, the antibody of the disclosure comprises a VH CDR2 inaccordance with the following Kabat amino acid sequences: SEQ ID NO:76,SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:103, SEQ ID NO:112, SEQ ID NO:121,SEQ ID NO:130, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:157, SEQ IDNO:166, SEQ ID NO:175, SEQ ID NO:184, SEQ ID NO:193, SEQ ID NO:202, orSEQ ID NO:211. In some embodiments, the antibody of the disclosurecomprises a VH CDR2 in accordance with the following Chothia amino acidsequences: SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:95, SEQ ID NO:104, SEQID NO:113, SEQ ID NO:122, SEQ ID NO:131, SEQ ID NO:140, SEQ ID NO:149,SEQ ID NO:158, SEQ ID NO:167, SEQ ID NO:176, SEQ ID NO:185, SEQ IDNO:194, SEQ ID NO:203, or SEQ ID NO:212. In some embodiments, theantibody of the disclosure comprises a VH CDR3 in accordance with thefollowing amino acid sequences: SEQ ID NO:78, SEQ ID NO:87, SEQ IDNO:96, SEQ ID NO:105, SEQ ID NO:114, SEQ ID NO:123, SEQ ID NO:132, SEQID NO:141, SEQ ID NO:150, SEQ ID NO:159, SEQ ID NO:168, SEQ ID NO:177,SEQ ID NO:186, SEQ ID NO:195, SEQ ID NO:204, or SEQ ID NO:213. In someembodiments, the antibody of the disclosure comprises a VL CDR1 inaccordance with the following amino acid sequences: SEQ ID NO:79, SEQ IDNO:88, SEQ ID NO:97, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:124, SEQ IDNO:133, SEQ ID NO:142, SEQ ID NO:151, SEQ ID NO:160, SEQ ID NO:169, SEQID NO:178, SEQ ID NO:187, SEQ ID NO:196, SEQ ID NO:205, SEQ ID NO:214,or SEQ ID NO:220. In some embodiments, the antibody of the disclosurecomprises a VL CDR2 in accordance with the following amino acidsequences: SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98, SEQ ID NO:107, SEQID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:143, SEQ ID NO:152,SEQ ID NO:161, SEQ ID NO:170, SEQ ID NO:179, SEQ ID NO:188, SEQ IDNO:197, SEQ ID NO:206, SEQ ID NO:215, or SEQ ID NO:221. In someembodiments, the antibody of the disclosure comprises a VL CDR3 inaccordance with the following amino acid sequences: SEQ ID NO:81, SEQ IDNO:90, SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:117, SEQ ID NO:126, SEQ IDNO:135, SEQ ID NO:144, SEQ ID NO:153, SEQ ID NO:162, SEQ ID NO:171, SEQID NO:180, SEQ ID NO:189, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:216,or SEQ ID NO:222. In some embodiments, the antibody of the disclosurecomprises the VH CDR1 in accordance with SEQ ID NO:217. In someembodiments, the antibody of the disclosure comprises the VH CDR2 inaccordance with SEQ ID NO:218. In some embodiments, the antibody of thedisclosure comprises the VH CDR3 in accordance with SEQ ID NO:219.

In certain embodiments, the antibody of the disclosure comprises the VHCDR1, CDR2, and CDR3 in accordance with Kabat, Chothia, or extendedsequences and/or VL CDR1, CDR2, and CDR3 in accordance with Kabat and/orChothia amino acid sequences of an antibody selected from: F4.7.6 VH,F4.7.8 VH, F5.1.11 VH, F5.1.9 VH, F4.7.062 VH, F5.1.11.01 VH, F5.1.11.02VH, F5.1.11.03 VH, F5.1.11.04 VH, F5.1.11.05 VH, F5.1.11.06 VH,F5.1.11.07 VH, F5.1.11.08 VH, F5.1.11.09 VH, F5.1.9.5 VH, d1C7 VH,F4.7.6 VL, F4.7.8 VL, F5.1.11 VL, F5.1.9 VL, F4.7.062 VL, F5.1.11.01 VL,F5.1.11.02 VL, F5.1.11.03 VL, F5.1.11.04 VL, F5.1.11.05 VL, F5.1.11.06VL, F5.1.11.07 VL, F5.1.11.08 VL, F5.1.11.09 VL, F5.1.9.5 VL, or d1C7 VLas shown in Table 7. In certain embodiments, the antibody of thedisclosure comprises the VH CDR1, CDR2, and CDR3 in accordance withKabat, Chothia, or extended sequences and/or VL CDR1, CDR2, and CDR3 inaccordance with Kabat and/or Chothia amino acid sequences of antibodyF5.1.11.02 as shown in Table 7.

A suitable portion or variant of any of these VH or VL sequences willretain the ability to bind to IL-2. In some embodiments, it willpreferably retain the ability to specifically bind to IL-2. In someembodiments, it will preferably retain the ability to specifically bindto the same or similar epitope or region of the IL-2 molecule as theantibody from which it is derived. In some embodiments, it willpreferably retain one or more additional functions of the antibody fromwhich it is derived, such as binding hIL-2, reducing hIL-2 binding toIL-2Rα and IL-2Rβ, and being Treg sparing, among others.

In some embodiments, a suitable portion or variant of any of these VH orVL sequences will retain the ability to bind to hIL-2. In someembodiments, it will retain the ability to specifically bind to hIL-2.In some embodiments, it will retain the ability to specifically bind tothe same or similar epitope or region of the hIL-2 molecule as theantibody from which it is derived. In some embodiments, it will retainone or more additional functions of the antibody from which it isderived, such as binding hIL-2, reducing hIL-2 binding to IL-2Rα andIL-2Rβ, and being Treg sparing, among others.

An antibody of the disclosure may comprise a CDR region from thespecific antibody identified herein such as a CDR region from within SEQID NO:1-32. In some embodiments, such an antibody will preferably retainthe ability to bind to IL-2 as described herein. In some embodiments,such an antibody will preferably retain the ability to bind to hIL-2 asdescribed herein. For example, the CDR sequences of the antibodies ofthe disclosure are shown in the Sequence Listing Table (Table 7) and theSEQ ID NOs. are shown in Table 6. In certain embodiments, an antibody ofthe disclosure comprises 1, 2, 3, 4, 5 or 6 CDRs from within an antibodyof the disclosure.

In one aspect, the disclosure provides an antibody variant comprising 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative ornon-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 additions and/or deletions to one or more of theCDRs listed above. In a further aspect, the variant shares at least 65%,at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity with one ormore of the CDR sequences listed above, and wherein the antibody orantigen-binding portion specifically binds IL-2. In some embodiments,the antibody or antigen-binding portion specifically binds hIL-2.

Polynucleotides, Vectors, and Host Cells

The disclosure also provides polynucleotides encoding any of theantibodies, including antibody portions and modified antibodiesdescribed herein, such as, e.g., antibodies having impaired effectorfunction. In another aspect, the disclosure provides a method of makingany of the polynucleotides described herein. Polynucleotides can be madeand expressed by procedures known in the art. Accordingly, thedisclosure provides polynucleotides or compositions, includingpharmaceutical compositions, comprising polynucleotides, encoding any ofthe following IL-2 antibodies and antigen-binding portions thereof:F4.7.6 VH, F4.7.8 VH, F5.1.11 VH, F5.1.9 VH, F4.7.062 VH, F5.1.11.01 VH,F5.1.11.02 VH, F5.1.11.03 VH, F5.1.11.04 VH, F5.1.11.05 VH, F5.1.11.06VH, F5.1.11.07 VH, F5.1.11.08 VH, F5.1.11.09 VH, F5.1.9.5 VH, d1C7 VH,F4.7.6 VL, F4.7.8 VL, F5.1.11 VL, F5.1.9 VL, F4.7.062 VL, F5.1.11.01 VL,F5.1.11.02 VL, F5.1.11.03 VL, F5.1.11.04 VL, F5.1.11.05 VL, F5.1.11.06VL, F5.1.11.07 VL, F5.1.11.08 VL, F5.1.11.09 VL, F5.1.9.5 VL, or d1C7 VLor any portion or part thereof having the ability to bind IL-2.

In one embodiment, the VH and VL domains, or antigen-binding portionthereof, or full length HC or LC, are encoded by separatepolynucleotides. Alternatively, both VH and VL, or antigen-bindingportion thereof, or HC and LC, are encoded by a single polynucleotide.

In another aspect, the disclosure provides polynucleotides and variantsthereof encoding an IL-2 antibody, wherein such variant polynucleotidesshare at least 70%, at least 75%, at least 80%, at least 85%, at least87%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to any of the specific nucleicacid disclosed herein. These amounts are not meant to be limiting, andincrements between the recited percentages are specifically envisionedas part of the disclosure.

Polynucleotides complementary to any such sequences are also encompassedby the present disclosure. Polynucleotides may be single-stranded(coding or antisense) or double-stranded, and may be DNA (genomic, cDNAor synthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present disclosure, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. In some embodiments, variants exhibit at least about 70%identity, in some embodiments, at least about 80% identity, in someembodiments, at least about 90% identity, and in some embodiments, atleast about 95% identity to a polynucleotide sequence that encodes anative antibody or a portion thereof. These amounts are not meant to belimiting, and increments between the recited percentages arespecifically envisioned as part of the disclosure.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegAlign® program in the Lasergene® suite of bioinformatics software(DNASTAR®, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

In some embodiments, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present disclosure.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present disclosure. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this disclosure can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors are further provided. Expression vectors generallyare replicable polynucleotide constructs that contain a polynucleotideaccording to the disclosure. It is implied that an expression vectormust be replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the polynucleotides of interest and/or thepolynucleotides themselves, can be introduced into the host cell by anyof a number of appropriate means, including electroporation,transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (e.g., where the vector is aninfectious agent such as vaccinia virus). The choice of introducingvectors or polynucleotides will often depend on features of the hostcell.

The disclosure also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). In some embodiments, the host cells express the cDNAs at alevel of about 5 fold higher, in some embodiments, 10 fold higher, andin some embodiments, 20 fold higher than that of the correspondingendogenous antibody or protein of interest, if present, in the hostcells. Screening the host cells for a specific binding to IL-2 iseffected by an immunoassay or FACS. A cell overexpressing the antibodyor protein of interest can be identified.

An expression vector can be used to direct expression of an IL-2antibody. One skilled in the art is familiar with administration ofexpression vectors to obtain expression of an exogenous protein in vivo.See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.Administration of expression vectors includes local or systemicadministration, including injection, oral administration, particle gunor catheterized administration, and topical administration. In anotherembodiment, the expression vector is administered directly to thesympathetic trunk or ganglion, or into a coronary artery, atrium,ventrical, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer, J. A.Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al.,J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA,1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy, 1994, 1:51;Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy,1995, 1:185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther., 1992, 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther., 1992,3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 1989,264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol., 1994, 14:2411, andin Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount, or “effective amount”, ofan IL-2 antibody, an antigen-binding portion, or an antibody:IL-2complex comprising the IL-2 antibody as described herein arecontemplated by the present disclosure. As used herein, a“therapeutically effective”, or “effective”, amount refers to an amountof an antibody or portion thereof that is of sufficient quantity toresult in a decrease in severity of disease symptoms, an increase infrequency and duration of disease symptom-free periods, or a preventionof impairment or disability due to the disease affliction—either as asingle dose or according to a multiple dose regimen, alone or incombination with other agents. One of ordinary skill in the art would beable to determine such amounts based on such factors as the subject'ssize, the severity of the subject's symptoms, and the particularcomposition or route of administration selected. The subject may be ahuman or non-human animal (e.g., rabbit, rat, mouse, monkey or otherlower-order primate).

An antibody, antigen-binding portion, or antibody:IL-2 complexcomprising the IL-2 antibody of the disclosure might be co-administeredwith known medicaments, and in some instances the antibody might itselfbe modified. For example, an antibody could be conjugated to animmunotoxin or radioisotope to potentially further increase efficacy.Regarding co-administration with additional therapeutic agents, suchagents can include a cytotoxic agent, a radiotoxic agent or animmunosuppressive agent. The antibody can be linked to the agent (as animmunocomplex) or can be administered separately from the agent. In thelatter case (separate administration), the antibody can be administeredbefore, after or concurrently with the agent or can be co-administeredwith other known therapies, e.g., an anti-cancer therapy, e.g.,radiation. Co-administration of the IL-2 antibodies, antigen-bindingportions thereof, or antibody:IL-2 complexes comprising the IL-2antibodies of the present disclosure with a therapeutic agent providestwo agents which operate via different mechanisms may provide atherapeutic and perhaps synergistic effect to human disease.

The antibodies, antigen-binding portions, and antibody:IL-2 complexescomprising the IL-2 antibodies disclosed herein can be used astherapeutics or diagnostic tools in a variety of situations where IL-2is undesirably active, such as inflammatory conditions such as anautoimmune disease, or situations where immunosuppression is desired. Incertain embodiments, immunosuppression therapy may be in preparation fororgan or bone marrow transplantation. Given the involvement of IL-2 ininflammatory pathways and in numerous diseases, disorders andconditions, many such diseases, disorders or conditions are particularlysuitable for treatment with an antibody, antigen-binding portion, or anantibody:IL-2 complex comprising the IL-2 antibody of the presentdisclosure. Accordingly, the IL-2 antibodies, antigen-binding portionsthereof, or antibody:IL-2 complexes comprising the IL-2 antibodies ofthis disclosure can be used in the treatment or prevention ofIL-2-mediated disorders or IL-2-deficiency disorders. In addition, thedisclosure provides for use of the IL-2 antibodies, antigen-bindingportions thereof, or antibody:IL-2 complexes comprising the IL-2antibodies of this disclosure in the manufacture of a medicament for usein treatment or prevention of IL-2-mediated disorders or IL-2-deficiencydisorders. In another embodiment, this application discloses IL-2antibodies, antigen-binding portions thereof, or antibody:IL-2 complexescomprising the IL-2 antibodies of this disclosure for use in treatmentof IL-2-mediated disorders or IL-2-deficiency disorders. In a furtherembodiment, this application discloses pharmaceutical compositionscomprising the IL-2 antibodies, antigen-binding portions thereof, orantibody:IL-2 complexes comprising the IL-2 antibodies of thisdisclosure for use in treating or preventing IL-2-mediated diseases orIL-2-deficiency disorders. In certain embodiments, the antibodyspecifically binds hIL-2.

In certain embodiments, these diseases include immunologic diseases,such as Graft vs Host disease. In certain embodiments, the disease maybe any disease associated with IL-2, such as muscular dystrophy andobesity. Exemplary autoimmune diseases and disorders that may be treatedwith the antibodies, antigen-binding portions thereof, or antibody:IL-2complexes comprising the IL-2 antibodies provided herein include, forexample, inflammatory responses such as inflammatory skin diseasesincluding psoriasis and dermatitis (e. g. atopic dermatitis);dermatomyositis; systemic scleroderma and sclerosis; responsesassociated with inflammatory bowel disease (such as Crohn's disease andulcerative colitis); respiratory distress syndrome (including adultrespiratory distress syndrome; and ARDS); dermatitis; meningitis;encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis;systemic lupus erythematosus (SLE); diabetes mellitus (e.g., Type Idiabetes mellitus); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; Wegener's disease; pernicious anemia (Addison's disease);diseases involving leukocyte diapedesis; central nervous system (CNS)inflammatory disorder; multiple organ injury syndrome; hemolytic anemia(including, but not limited to cryoglobinemia or Coombs positiveanemia); myasthenia gravis; antigen-antibody complex mediated diseases;anti-glomerular basement membrane disease; antiphospholipid syndrome;allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies;vitiligo; Reiter's disease; stiff-man syndrome; Behcet's disease; giantcell arteritis; immune complex nephritis; IgA nephropathy; IgMpolyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmunethrombocytopenia and autoimmune hemolytic diseases; Hashimoto'sthyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmunelymphoproliferative syndrome (ALPS); autoimmune uveoretinitis;Guillain-Barre syndrome; Goodpasture's syndrome; mixed connective tissuedisease; autoimmune-associated infertility; polyarteritis nodosa;alopecia areata; and idiopathic myxedema. In some embodiments, thecondition that can be treated with the present compositions and methodsis diabetes mellitus (e.g., Type I diabetes mellitus). In someembodiments, the disease is Type I diabetes mellitus. In someembodiments, the disease is juvenile onset diabetes.

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the present disclosure may be formulated in aconventional manner using one or more pharmaceutically acceptablecarriers or excipients and administered as more fully discussed below.

Determining a therapeutically effective amount of an antibody,antigen-binding portion thereof, or antibody:IL-2 complex comprising theIL-2 antibody of this disclosure according to the present disclosurewill largely depend on particular patient characteristics, route ofadministration, and the nature of the disorder being treated and is morefully discussed below.

Administration and dosing of the antibody, antigen-binding portionthereof, or antibody:IL-2 complex comprising the IL-2 antibody of thisdisclosure are more fully discussed elsewhere below.

Diagnostic Methods

The IL-2 antibodies, or antigen-binding portions thereof disclosedherein can be used for diagnostic testing and imaging. For example, theIL-2 antibodies or antigen-binding portions thereof can be used in anELISA assay. The antibodies or antigen-binding portions thereof can alsobe used as a radiolabeled monoclonal antibody. See, for example,Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging AndTherapy, Plenum Press (1988); Chase, “Medical Applications ofRadioisotopes,” in Remington's Pharmaceutical Sciences, 18th Edition,Gennaro et al. (eds.), Mack Publishing Co., pp. 624-652 (1990); andBrown, “Clinical Use of Monoclonal Antibodies,” in Biotechnology andPharmacy, Pezzuto et al. (eds.), Chapman and Hall, pp. 227-249 (1993);Grossman, 1986, Urol. Clin. North Amer. 13:465-474; Unger et al., 1985,Invest. Radiol. 20:693-700; and Khaw et al., 1980, Science 209:295-297.This technique, also known as immunoscintigraphy, uses a gamma camera todetect the location of gamma-emitting radioisotopes conjugated tomonoclonal antibodies. Diagnostic imaging can be used to diagnosecancer, autoimmune disease, infectious disease and/or cardiovasculardisease. (See, e.g., Brown, supra.)

In one embodiment, the IL-2 antibodies or antigen-binding portionsthereof can be used to diagnose IL-2-related diseases, disorders, orconditions, including immune-related diseases. For example, theantibodies, or antigen-binding portions thereof, can be used to detectIL-2 levels in patients, among other uses.

In addition to diagnosis, the IL-2 antibodies or antigen-bindingportions thereof can be used to monitor therapeutic responses, detectrecurrences of a disease, and guide subsequent clinical decisions.

In some embodiments, for diagnostic and monitoring purposes,radioisotopes may be bound to antibody portions either directly orindirectly by using an intermediary functional group. Such intermediaryfunctional groups include, for example, DTPA(diethylenetriaminepentaacetic acid) and EDTA (ethylene diaminetetraacetic acid). The radiation dose delivered to the patient istypically maintained at as low a level as possible. This may beaccomplished through the choice of isotope for the best combination ofminimum half-life, minimum retention in the body, and minimum quantityof isotope which will permit detection and accurate measurement.Examples of radioisotopes which can be bound to antibodies and areappropriate for diagnostic imaging include ⁹⁹mTc and ¹¹¹In.

Studies indicate that antibody portions, particularly Fab and Fab′,provide suitable tumor/background ratios. (See, e.g., Brown, supra.)

The IL-2 antibody or antigen-binding portions thereof also can belabeled with paramagnetic ions for purposes of in vivo diagnosis.Elements which are particularly useful for Magnetic Resonance Imaginginclude Gd, Mn, Dy, and Fe ions.

The IL-2 antibody or antigen-binding portions thereof can also detectthe presence of IL-2 in vitro. In such immunoassays, the antibody orantigen-binding portions thereof may be utilized in liquid phase orbound to a solid-phase carrier. For example, an intact antibody, orantigen-binding portion thereof, can be attached to a polymer, such asaminodextran, in order to link the antibody component to an insolublesupport such as a polymer-coated bead, plate, or tube. In someembodiments, the IL-2 for detection is human IL-2 (hIL-2). In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 0.1 ng/mL and 1000 ng/mL in vitro. In some embodiments, thehIL-2 for detection is present at a concentration of between 0.1 ng/mLand 750 ng/mL in vitro. In some embodiments, the hIL-2 for detection ispresent at a concentration of between 0.5 ng/mL and 500 ng/mL in vitro.In some embodiments, the hIL-2 for detection is present at aconcentration of between 0.5 ng/mL and 250 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 0.5 ng/mL and 100 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 0.5 ng/mL and 50ng/mL in vitro. In some embodiments, the hIL-2 for detection is presentat a concentration of between 0.5 ng/mL and 25 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 0.5 ng/mL and 10 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 0.5 ng/mL and 5ng/mL in vitro. In some embodiments, the hIL-2 for detection is presentat a concentration of between 0.5 ng/mL and 1 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 0.1 ng/mL and 5 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 0.1 ng/mL and 1ng/mL in vitro. In some embodiments, the hIL-2 for detection is presentat a concentration of between 0.8 ng/mL and 500 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 1 ng/mL and 500 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 1 ng/mL and 250ng/mL in vitro. In some embodiments, the hIL-2 for detection is presentat a concentration of between 1 ng/mL and 100 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 1 ng/mL and 50 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 1 ng/mL and 25ng/mL in vitro. In some embodiments, the hIL-2 for detection is presentat a concentration of between 1 ng/mL and 10 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 50 ng/mL and 500 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 100 ng/mL and 500ng/mL in vitro. In some embodiments, the hIL-2 for detection is presentat a concentration of between 250 ng/mL and 500 ng/mL in vitro. In someembodiments, the hIL-2 for detection is present at a concentration ofbetween 400 ng/mL and 500 ng/mL in vitro. In some embodiments, the hIL-2for detection is present at a concentration of between 500 ng/mL and1000 ng/mL in vitro. In some embodiments, the hIL-2 for detection ispresent at a concentration of between 750 ng/mL and 1000 ng/mL in vitro.In some embodiments, the hIL-2 for detection is present at aconcentration of 0.8 ng/mL in vitro. In some embodiments, the hIL-2 fordetection is present at a concentration of 500 ng/mL in vitro. Theseamounts are not meant to be limiting, and increments between the recitedvalues are specifically envisioned as part of the disclosure.

Alternatively, the IL-2 antibody or antigen-binding portions thereof canbe used to detect the presence of particular antigens in tissue sectionsprepared from a histological specimen. Such in situ detection can beaccomplished, for example, by applying a detectably-labeled IL-2antibody or antigen-binding portion thereof to the tissue sections. Insitu detection can be used to determine the presence of a particularantigen and to determine the distribution of the antigen in the examinedtissue. General techniques of in situ detection are well known to thoseof ordinary skill. (See, e.g., Ponder, “Cell Marking Techniques andTheir Application,” in Mammalian Development: A Practical Approach, Monk(ed.), IRL Press, pp. 115-138 (1987); Coligan et al., supra.)

Detectable labels such as enzymes, fluorescent compounds, electrontransfer agents, and the like can be linked to a carrier by conventionalmethods well known to the art. These labeled carriers and the antibodyconjugates prepared from them can be used for in vitro immunoassays andfor in situ detection, much as an antibody conjugate can be prepared bydirect attachment of the labels to antibody. The loading of the antibodyconjugates with a plurality of labels can increase the sensitivity ofimmunoassays or histological procedures, where only a low extent ofbinding of the antibody, or antibody portion, to target antigen isachieved.

Compositions

The disclosure also provides pharmaceutical compositions comprising aneffective amount of an IL-2 antibody described herein. Examples of suchcompositions, as well as how to formulate, are also described herein. Insome embodiments, the composition comprises one or more IL-2 antibodies.In other embodiments, the IL-2 antibody recognizes IL-2. In otherembodiments, the IL-2 antibody is a human antibody. In otherembodiments, the IL-2 antibody is a humanized antibody. In someembodiments, the IL-2 antibody comprises a constant region that iscapable of triggering a desired immune response, such asantibody-mediated lysis or ADCC. In other embodiments, the IL-2 antibodycomprises a constant region that does not trigger an unwanted orundesirable immune response, such as antibody-mediated lysis or ADCC. Inother embodiments, the IL-2 antibody comprises one or more CDR(s) of theantibody (such as one, two, three, four, five, or, in some embodiments,all six CDRs).

It is understood that the compositions can comprise more than one IL-2antibody (e.g., a mixture of IL-2 antibodies that recognize differentepitopes of IL-2). Other exemplary compositions comprise more than oneIL-2 antibody that recognize the same epitope(s), or different speciesof IL-2 antibodies that bind to different epitopes of IL-2. In someembodiments, the compositions comprise a mixture of IL-2 antibodies thatrecognize different variants of IL-2.

The composition used in the present disclosure can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and practice of Pharmacy 20th Ed., 2000,Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations, and may comprise buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

The IL-2 antibody and compositions thereof can also be used inconjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents.

In certain embodiments, the IL-2 antibody is complexed with IL-2 beforeadministration. In certain embodiments, the IL-2 antibody is notcomplexed with IL-2 before administration.

The disclosure also provides compositions, including pharmaceuticalcompositions, comprising any of the polynucleotides of the disclosure.In some embodiments, the composition comprises an expression vectorcomprising a polynucleotide encoding the antibody as described herein.In other embodiments, the composition comprises an expression vectorcomprising a polynucleotide encoding any of the antibodies describedherein. In still other embodiments, the composition comprises either orboth of the polynucleotides encoding the sequence shown in SEQ ID NO: 1and SEQ ID NO: 2, either or both of the polynucleotides encoding thesequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, either or both of thepolynucleotides encoding the sequence shown in SEQ ID NO:5 and SEQ IDNO:6, either or both of the polynucleotides encoding the sequence shownin SEQ ID NO:7 and SEQ ID NO:8, either or both of the polynucleotidesencoding the sequence shown in SEQ ID NO:9 and SEQ ID NO:10, either orboth of the polynucleotides encoding the sequence shown in SEQ ID NO:11and SEQ ID NO:12, either or both of the polynucleotides encoding thesequence shown in SEQ ID NO:13 and SEQ ID NO:14, either or both of thepolynucleotides encoding the sequence shown in SEQ ID NO:15 and SEQ IDNO:16, either or both of the polynucleotides encoding the sequence shownin SEQ ID NO:17 and SEQ ID NO:18, either or both of the polynucleotidesencoding the sequence shown in SEQ ID NO:19 and SEQ ID NO:20, either orboth of the polynucleotides encoding the sequence shown in SEQ ID NO:21and SEQ ID NO:22, either or both of the polynucleotides encoding thesequence shown in SEQ ID NO:23 and SEQ ID NO:24, either or both of thepolynucleotides encoding the sequence shown in SEQ ID NO:25 and SEQ IDNO:26, either or both of the polynucleotides encoding the sequence shownin SEQ ID NO:27 and SEQ ID NO:28, either or both of the polynucleotidesencoding the sequence shown in SEQ ID NO:29 and SEQ ID NO:30, or eitheror both of the polynucleotides encoding the sequence shown in SEQ IDNO:31 and SEQ ID NO:32. In still other embodiments, the compositioncomprises either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 36 and SEQ ID NO: 37, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 38 and SEQID NO: 39, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 40 and SEQ ID NO: 41, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 42 and SEQID NO: 43, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 44 and SEQ ID NO: 45, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 46 and SEQID NO: 47, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 48 and SEQ ID NO: 49, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 50 and SEQID NO: 51, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 52 and SEQ ID NO: 53, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 54 and SEQID NO: 55, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 56 and SEQ ID NO: 57, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 58 and SEQID NO: 59, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 60 and SEQ ID NO: 61, either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 62 and SEQID NO: 63, either or both of the polynucleotides comprising the sequenceshown in SEQ ID NO: 64 and SEQ ID NO: 65, or either or both of thepolynucleotides comprising the sequence shown in SEQ ID NO: 66 and SEQID NO: 67.

In another aspect, the polynucleotide can encode the VH, VL and/or bothVH and VL of the antibody of the disclosure. That is, the compositioncomprises a single polynucleotide or more than one polynucleotideencoding the antibody, or antigen-binding portion thereof, or thedisclosure.

Pharmaceutical compositions of the disclosure also can be administeredin combination therapy, such as, combined with other agents. Forexample, the combination therapy can include IL-2 antibody, orantigen-binding portion thereof, of the present disclosure combined withat least one other therapy wherein the therapy may be surgery,immunotherapy, or drug therapy.

The pharmaceutical compounds of the disclosure may include one or morepharmaceutically acceptable salts. Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will besuitable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

A pharmaceutical composition of the present disclosure may be prepared,packaged, or sold in a formulation suitable for ophthalmicadministration. Such formulations may, for example, be in the form ofeye drops including, for example, a 0.1%-1.0% (w/w) solution orsuspension of the active ingredient in an aqueous or oily liquidcarrier. Such drops may further comprise buffering agents, salts, or oneor more other of the additional ingredients described herein. Otherophthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form or in aliposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the disclosure are knownin the art and described, for example in Remington's PharmaceuticalSciences, Genaro, ed., Mack Publishing Co., Easton, Pa. (1985), which isincorporated herein by reference.

In one embodiment, the IL-2 antibody, or antigen-binding portionthereof, is administered in an intravenous formulation as a sterileaqueous solution containing 5 mg/mL, or in some embodiments, about 10mg/mL, or in some embodiments, about 15 mg/mL, or in some embodiments,about 20 mg/mL of antibody, with sodium acetate, polysorbate 80, andsodium chloride at a pH ranging from about 5 to 6. In some embodiments,the intravenous formulation is a sterile aqueous solution containing 5or 10 mg/mL of antibody, with 20 mM sodium acetate, 0.2 mg/mLpolysorbate 80, and 140 mM sodium chloride at pH 5.5. Further, asolution comprising an antibody, or antigen-binding portion thereof, cancomprise, among many other compounds, histidine, mannitol, sucrose,trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, and anycombination thereof, and many other compounds known in the relevant art.

In one embodiment, a pharmaceutical composition of the presentdisclosure comprises the following components: 100 mg IL-2 antibody orantigen-binding portion of the present disclosure, 10 mM histidine, 5%sucrose, and 0.01% polysorbate 80 at pH 5.8. This composition may beprovided as a lyophilized powder. When the powder is reconstituted atfull volume, the composition retains the same formulation.Alternatively, the powder may be reconstituted at half volume, in whichcase the composition comprises 100 mg IL-2 antibody or antigen-bindingportion thereof of the present disclosure, 20 mM histidine, 10% sucrose,and 0.02% polysorbate 80 at pH 5.8.

In one embodiment, part of the dose is administered by an intravenousbolus and the rest by infusion of the antibody formulation. For example,a 0.01 mg/kg intravenous injection of the IL-2 antibody, orantigen-binding portion thereof, may be given as a bolus, and the restof the antibody dose may be administered by intravenous injection. Apredetermined dose of the IL-2 antibody, or antigen-binding portionthereof, may be administered, for example, over a period of an hour anda half to two hours to five hours.

With regard to a therapeutic agent, where the agent is, e.g., a smallmolecule, it can be present in a pharmaceutical composition in the formof a physiologically acceptable ester or salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

In one embodiment the compositions of the disclosure are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released when the microorganisms are broken downor die. Pyrogenic substances also include fever-inducing, thermostablesubstances (glycoproteins) from the outer membrane of bacteria and othermicroorganisms. Both of these substances can cause fever, hypotensionand shock if administered to humans. Due to the potential harmfuleffects, it is advantageous to remove even low amounts of endotoxinsfrom intravenously administered pharmaceutical drug solutions. The Foodand Drug Administration (“FDA”) has set an upper limit of 5 endotoxinunits (EU) per dose per kilogram body weight in a single one hour periodfor intravenous drug applications (The United States PharmacopeialConvention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeuticproteins are administered in amounts of several hundred or thousandmilligrams per kilogram body weight it is advantageous to remove eventrace amounts of endotoxin. In one embodiment, endotoxin and pyrogenlevels in the composition are less than 10 EU/mg, or less than 5 EU/mg,or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg,or less than 0.001 EU/mg. In another embodiment, endotoxin and pyrogenlevels in the composition are less than about 10 EU/mg, or less thanabout 5 EU/mg, or less than about 1 EU/mg, or less than about 0.1 EU/mg,or less than about 0.01 EU/mg, or less than about 0.001 EU/mg.

In one embodiment, the disclosure comprises administering a compositionwherein said administration is oral, parenteral, intramuscular,intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal,intravenous, cutaneous, subcutaneous or transdermal.

In another embodiment the disclosure further comprises administering acomposition in combination with other therapies, such as surgery,chemotherapy, hormonal therapy, biological therapy, immunotherapy orradiation therapy.

Dosing/Administration

To prepare pharmaceutical or sterile compositions including an IL-2antibody, or antigen-binding portion thereof of the disclosure, theantibody is mixed with a pharmaceutically acceptable carrier orexcipient. Formulations of therapeutic and diagnostic agents can beprepared by mixing with physiologically acceptable carriers, excipients,or stabilizers in the form of, e.g., lyophilized powders, slurries,aqueous solutions, lotions, or suspensions (see, e.g., Hardman, et al.(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y.).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainembodiments, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak,1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis,Marcel Dekker, New York, N.Y.; Bach (ed.), 1993, Monoclonal Antibodiesand Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York,N.Y.; Baert, et al., 2003, New Engl. J. Med. 348:601-608; Milgrom, etal., 1999, New Engl. J. Med. 341:1966-1973; Slamon, et al., 2001, NewEngl. J. Med. 344:783-792; Beniaminovitz, et al., 2000, New Engl. J.Med. 342:613-619; Ghosh, et al., 2003, New Engl. J. Med. 348:24-32;Lipsky, et al., 2000, New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

Compositions comprising IL-2 antibodies or antigen-binding portionsthereof, of the disclosure can be provided by continuous infusion, or bydoses at intervals of, e.g., one day, one week, or 1-7 times per week.Doses may be provided intravenously, subcutaneously, topically, orally,nasally, rectally, intramuscular, intracerebrally, or by inhalation. Aspecific dose protocol is one involving the maximal dose or dosefrequency that avoids significant undesirable side effects. A totalweekly dose may be at least 0.05 μg/kg body weight, at least 0.2 μg/kg,at least 0.5 μg/kg, at least 1 μg/kg, at least 10 μg/kg, at least 100μg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, atleast 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg,or at least 50 mg/kg (see, e.g., Yang, et al., 2003, New Engl. J. Med.349:427-434; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698; Liu,et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, etal., 2003, Cancer. Immunol. Immunother. 52: 133-144). The dose may be atleast 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, atleast 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg.The doses administered to a subject may number at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12, or more.

For IL-2 antibodies or antigen-binding portions thereof of thedisclosure, the dosage administered to a patient may be 0.0001 mg/kg to100 mg/kg of the patient's body weight. The dosage may be between 0.0001mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg,0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg,0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or0.01 to 0.10 mg/kg of the patient's body weight.

The dosage of the IL-2 antibody or antigen-binding portion thereof maybe calculated using the patient's weight in kilograms (kg) multiplied bythe dose to be administered in mg/kg. The dosage of the antibodies ofthe disclosure may be 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg orless, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 μg/kg orless, 75 μg/kg or less, 70 μg/kg or less, 65 μg/kg or less, 60 μg/kg orless, 55 μg/kg or less, 50 μg/kg or less, 45 μg/kg or less, 40 μg/kg orless, 35 μg/kg or less, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg orless, 15 μg/kg or less, 10 μg/kg or less, 5 μg/kg or less, 2.5 μg/kg orless, 2 μg/kg or less, 1.5 μg/kg or less, 1 μg/kg or less, 0.5 μg/kg orless, or 0.1 μg/kg or less of a patient's body weight.

Unit dose of the IL-2 antibodies or antigen-binding portions thereof ofthe disclosure may be 0.1 mg to 200 mg, 0.1 mg to 175 mg, 0.1 mg to 150mg, 0.1 mg to 125 mg, 0.1 mg to 100 mg, 0.1 mg to 75 mg, 0.1 mg to 50mg, 0.1 mg to 30 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg,0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg,0.25 to 8 mg, 0.25 mg to 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mgto 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

The dosage of the IL-2 antibodies or antigen-binding portions thereof ofthe disclosure may achieve a serum titer of at least 0.1 μg/mL, at least0.5 μg/mL, at least 1 μg/mL, at least 2 μg/mL, at least 5 μg/mL, atleast 6 μg/mL, at least 10 μg/mL, at least μg/mL, at least 20 μg/mL, atleast 25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 125μg/mL, at least 150 μg/mL, at least 175 μg/mL, at least 200 μg/mL, atleast 225 μg/mL, at least 250 μg/mL, at least 275 μg/mL, at least 300μg/mL, at least 325 μg/mL, at least 350 μg/mL, at least 375 μg/mL, or atleast 400 μg/mL in a subject. Alternatively, the dosage of theantibodies of the disclosure may achieve a serum titer of at least 0.1μg/mL, at least 0.5 μg/mL, at least 1 μg/mL, at least, 2 μg/mL, at least5 μg/mL, at least 6 μg/mL, at least 10 μg/mL, at least 15 μg/mL, atleast 20 μg/mL, at least μg/mL, at least 50 μg/mL, at least 100 μg/mL,at least 125 μg/mL, at least 150 μg/mL, at least 175 μg/mL, at least 200μg/mL, at least 225 μg/mL, at least 250 μg/mL, at least 275 μg/mL, atleast 300 μg/mL, at least 325 μg/mL, at least 350 μg/mL, at least 375μg/mL, or at least 400 μg/mL in the subject.

Doses of IL-2 antibodies, or antigen-binding portions thereof of thedisclosure may be repeated and the administrations may be separated byat least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45days, 2 months, 75 days, 3 months, or at least 6 months.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside effects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent, 2001,Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or by sustained release systems or animplant (see, e.g., Sidman et al., 1983, Biopolymers 22:547-556; Langer,et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem.Tech. 12:98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA82:3688-3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA77:4030-4034; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary,the composition may also include a solubilizing agent and a localanesthetic such as lidocaine to ease pain at the site of the injection.In addition, pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,each of which is incorporated herein by reference their entirety. In oneembodiment, the IL-2 antibody, or antigen-binding portion thereof, or acomposition of the disclosure is administered using Alkermes AIR™pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

A composition of the present disclosure may also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Selected routes of administration for antibodies of thedisclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Parenteraladministration may represent modes of administration other than enteraland topical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion. Alternatively, a composition of the disclosure can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

If the IL-2 antibodies, or antigen-binding portions thereof, of thedisclosure are administered in a controlled release or sustained releasesystem, a pump may be used to achieve controlled or sustained release(see, Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20;Buchwald et al., 1980, Surgery 88:501; Saudek et al., 1989, N. Engl. J.Med. 321:514).

Polymeric materials can be used to achieve controlled or sustainedrelease of the therapies of the disclosure (see e.g., MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J., Macromol. ScL Rev. Macromol. Chem. 23:61;see also Levy et al, 1985, Science 11 225:190; During et al., 19Z9, Ann.Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71: 105); U.S. Pat.No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO99/15154; and PCT Publication No. WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),polyvinyl alcohol), polyacrylamide, polyethylene glycol), polylactides(PLA), polyoeactide-co-glycolides) (PLGA), and polyorthoesters. In oneembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. A controlled or sustained release system can be placed inproximity of the prophylactic or therapeutic target, thus requiring onlya fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer, 1990,Science 249:1527-1533. Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore antibodies of the disclosure or conjugates thereof. See, e.g., U.S.Pat. No. 4,526,938, International Patent Publication Nos. WO 91/05548,WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of aHuman Colon Cancer Xenograft Using a Sustained-Release Gel,”Radiotherapy and Oncology 59:179-189, Song et al., 1995, “AntibodyMediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal ofPharmaceutical Science and Technology 50:372-397, Cleek et ah, 1997,“Biodegradable Polymeric Carriers for a bFGF Antibody for CardiovascularApplication,” Pro. MI. Symp. Control. Rel. Bioact. Mater. 24:853-854,and Lam et al., 1997, “Microencapsulation of Recombinant HumanizedMonoclonal Antibody for Local Delivery,” Proc. MI. Symp. Control Rel.Bioact. Mater. 24:759-160, each of which is incorporated herein byreference in their entirety.

If the IL-2 antibody, or antigen-binding portion thereof, of thedisclosure is administered topically, it can be formulated in the formof an ointment, cream, transdermal patch, lotion, gel, shampoo, spray,aerosol, solution, emulsion, or other form well-known to one of skill inthe art. See, e.g., Remington's Pharmaceutical Sciences and Introductionto Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity, in someinstances, greater than water are typically employed. Suitableformulations include, without limitation, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like,which are, if desired, sterilized or mixed with auxiliary agents (e.g.,preservatives, stabilizers, wetting agents, buffers, or salts) forinfluencing various properties, such as, for example, osmotic pressure.Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, in some instances, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asfreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

If the compositions comprising IL-2 antibodies, or antigen-bindingportions thereof, are administered intranasally, it can be formulated inan aerosol form, spray, mist or in the form of drops. In particular,prophylactic or therapeutic agents for use according to the presentdisclosure can be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebuliser, with the use of asuitable propellant (e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas). In the case of a pressurized aerosol the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges (composed of, e.g., gelatin) for use in aninhaler or insufflator may be formulated containing a powder mix of thecompound and a suitable powder base such as lactose or starch.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art (see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10 th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001)Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams and Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams andWilkins, Phila., Pa.). An effective amount of therapeutic may decreasethe symptoms by at least 10 percent; by at least 20 percent; at leastabout 30 percent; at least 40 percent, or at least 50 percent.

Additional therapies (e.g., prophylactic or therapeutic agents), whichcan be administered in combination with the IL-2 antibodies, orantigen-binding portions of the disclosure, may be administered lessthan 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about1 hour apart, at about 1 to about 2 hours apart, at about 2 hours toabout 3 hours apart, at about 3 hours to about 4 hours apart, at about 4hours to about 5 hours apart, at about 5 hours to about 6 hours apart,at about 6 hours to about 7 hours apart, at about 7 hours to about 8hours apart, at about 8 hours to about 9 hours apart, at about 9 hoursto about 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours apart from the antibodies ofthe disclosure. The two or more therapies may be administered within onesame patient visit.

The IL-2 antibodies, or antigen-binding portions thereof, of thedisclosure and the other therapies may be cyclically administered.Cycling therapy involves the administration of a first therapy (e.g., afirst prophylactic or therapeutic agent) for a period of time, followedby the administration of a second therapy (e.g., a second prophylacticor therapeutic agent) for a period of time, optionally, followed by theadministration of a third therapy (e.g., prophylactic or therapeuticagent) for a period of time and so forth, and repeating this sequentialadministration, i.e., the cycle in order to reduce the development ofresistance to one of the therapies, to avoid or reduce the side effectsof one of the therapies, and/or to improve the efficacy of thetherapies.

In one embodiment, the IL-2 antibodies of the disclosure can beco-administered with compositions for treating autoimmune diseases anddisorders, including, but not limited to, adriamycin, azathiopurine,busulfan, cyclophosphamide, cyclosporine A, Cytoxan, fludarabine,5-fluorouracil, methotrexate, mycophenolate mofetil, 6-mercaptopurine, acorticosteroid, a nonsteroidal anti-inflammatory, sirolimus (rapamycin),and tacrolimus (FK-506). In alternative embodiments, theimmunomodulatory or immunosuppressive agent is an antibody selected fromthe group consisting of muromonab-CD3, alemtuzumab (Campath®),basiliximab, daclizumab, muromonab (OKT3®), rituximab, anti-thymocyteglobulin and IVIg, and others, which are known to persons skilled in theart.

In one embodiment, the IL-2 antibodies of the disclosure can beco-administered with compositions for treating diabetes, including, butnot limited to, biguanides (e.g., buformin, metformin, and phenform),hormones and analogs thereof (amylin, insulin, insulin aspart, insulindetemir, insulin glargine, insulin glulisine, insulin lispro,liraglutide, and pramlintide), sulfonylurea derivatives (acetohexamide,carbutamide, chlorpropamide, glibornuride, gliclazide, glimepiride,glipizide, gliquidone, glisoxepid, glyburide, glybuthiazole, glybuzole,glyhexamide, glymidine, tolazamide, tolbutamide, and tolcyclamide),thiazolidinediones (pioglitazone, rosiglitazone, and troglitazone),acarbose, exenatide, miglitol, mitiglinide, muraglitazar, nateglinide,repaglinide, sitagliptin, tesaglitazar, vildagliptin, and voglibose.

In certain embodiments, the IL-2 antibodies, or antigen-binding portionsthereof of the disclosure can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the disclosure cross the BBB (if desired), theycan be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;and 5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade, 1989, J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman etal., 1995, FEBS Lett. 357: 140; M. Owais et al., 1995, Antimicrob.Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe etal. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreier et al. (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen, 1994, FEBSLett. 346:123; Killion; Fidler, 1994; Immunomethods 4:273.

The disclosure provides protocols for the administration ofpharmaceutical composition comprising IL-2 antibodies, orantigen-binding portions thereof, of the disclosure alone or incombination with other therapies to a subject in need thereof. Thetherapies (e.g., prophylactic or therapeutic agents) of the combinationtherapies of the present disclosure can be administered concomitantly orsequentially to a subject. The therapy (e.g., prophylactic ortherapeutic agents) of the combination therapies of the presentdisclosure can also be cyclically administered. Cycling therapy involvesthe administration of a first therapy (e.g., a first prophylactic ortherapeutic agent) for a period of time, followed by the administrationof a second therapy (e.g., a second prophylactic or therapeutic agent)for a period of time and repeating this sequential administration, i.e.,the cycle, in order to reduce the development of resistance to one ofthe therapies (e.g., agents) to avoid or reduce the side effects of oneof the therapies (e.g., agents), and/or to improve, the efficacy of thetherapies.

The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the disclosure can be administered to a subjectconcurrently. The term “concurrently” is not limited to theadministration of therapies (e.g., prophylactic or therapeutic agents)at exactly the same time, but rather it is meant that a pharmaceuticalcomposition comprising IL-2 antibodies, or antigen-binding portionsthereof, of the disclosure are administered to a subject in a sequenceand within a time interval such that the antibodies of the disclosure orconjugates thereof can act together with the other therapy(ies) toprovide an increased benefit than if they were administered otherwise.For example, each therapy may be administered to a subject at the sametime or sequentially in any order at different points in time; however,if not administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeutic orprophylactic effect. Each therapy can be administered to a subjectseparately, in any appropriate form and by any suitable route. Invarious embodiments, the therapies (e.g., prophylactic or therapeuticagents) are administered to a subject less than 15 minutes, less than 30minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hourto about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 weekapart. In other embodiments, two or more therapies (e.g., prophylacticor therapeutic agents) are administered to a within the same patientvisit.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

Kits

The disclosure also provides kits comprising any or all of theantibodies described herein. Kits of the disclosure include one or morecontainers comprising an IL-2 antibody described herein and instructionsfor use in accordance with any of the methods of the disclosuredescribed herein. Generally, these instructions comprise a descriptionof administration of the antibody for the above described therapeutictreatments. In some embodiments, kits are provided for producing asingle-dose administration unit. In certain embodiments, the kit cancontain both a first container having a dried protein and a secondcontainer having an aqueous formulation. In certain embodiments, kitscontaining an applicator, e.g., single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes), are included.

The instructions relating to the use of an IL-2 antibody generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the disclosure are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) are alsoacceptable.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an IL-2 antibody of thedisclosure. The container may further comprise a second pharmaceuticallyactive agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The disclosure also provides diagnostic kits comprising any or all ofthe antibodies described herein. The diagnostic kits are useful for, forexample, detecting the presence of IL-2 in a sample. In someembodiments, a diagnostic kit can be used to identify an individual witha latent disease, disorder or condition that may put them at risk ofdeveloping IL-2-mediated disease, disorder or condition or an IL-2deficiency disease, disorder or condition. In some embodiments, adiagnostic kit can be used to detect the presence and/or level of IL-2in an individual suspected of having an IL-2 mediated disease or an IL-2deficiency disease, disorder or condition.

Diagnostic kits of the disclosure include one or more containerscomprising an IL-2 antibody described herein and instructions for use inaccordance with any of the methods of the disclosure described herein.Generally, these instructions comprise a description of use of the IL-2antibody to detect the presence of IL-2 in individuals at risk for, orsuspected of having, an IL-2 mediated disease or an IL-2 deficiencydisease, disorder or condition. In some embodiments, an exemplarydiagnostic kit can be configured to contain reagents such as, forexample, an IL-2 antibody, a negative control sample, a positive controlsample, and directions for using the kit.

EQUIVALENTS

The foregoing description and following Examples detail certain specificembodiments of the disclosure and describes the best mode contemplatedby the inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the disclosure may bepracticed in many ways and the disclosure should be construed inaccordance with the appended claims and any equivalents thereof.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed disclosure below.The following examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

EXEMPLARY EMBODIMENTS

The disclosure is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the disclosure should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

EXAMPLES Example 1. IL-2 Antibody Binding Kinetics and Affinity

The affinity of the antibodies for IL-2 was determined by surfaceplasmon resonance using a Biacore T200 instrument (GE Healthcare,Piscataway, N.J.). Anti-IL-2 IgGs were captured on a CM5 sensor chip byan anti-human IgG prepared using the Biacore Human Antibody Capture Kitaccording to the manufactures directions (GE Healthcare). The mouseanti-human IL-2 clone 5344 (clone 5344.111, BD Biosciences) was capturedon an anti-mouse IgG capture surface prepared using the Biacore MouseAntibody Capture Kit according to the manufactures directions (GEHealthcare). Experiments were performed at 25° C. using a 30 μL/minuteflow rate in 0.01 M HEPES pH 7.4, 0.15 M NaCl and 0.005% v/v surfactantP20 (HBS-P) buffer. After each cycle, the chip surface was regeneratedwith 3 M MgCl₂ and new antibody captured. Recombinant IL-2 (Humanzyme,Chicago, Ill.) was injected over the surface for 3 minutes and theassociation monitored for a further 20 minutes. Data were analyzed usingthe Biacore T200 Evaluation software, the signal from the adjacentcontrol flow cell with only the capture antibody immobilized wasbackground subtracted along with buffer only injections for eachantibody. A 1:1 Langmuir binding model was used to fit all bindingcurves.

Results

The human antibodies tested bind to IL-2 with affinities ranging from536 pM to 13.4 nM (Table 3). The antibodies identified for their abilityto inhibit IL-2Rβ binding and decrease binding to IL-2Rα relative to thed1C7/IL-2 complex, have weaker affinities for IL-2 compared to thecomparator clones 16C3 and d1C7. The 5344 antibody was found to have avery stable off-rate beyond the detection limits of the Biacoreinstrument, the calculated affinity is assumed to be 50 pM or less, witha significantly slower off-rate compared to the human antibody clonestested. The significantly slower off-rate of 5344 may contribute to theantibody not having the desired Treg sparing properties exhibited byother hIL-2 antibodies described in this application below (Example 5).

TABLE 3 Clone k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) F4.7.062 3.77E+065.80E−03 1.54E−09 F4.7.6 2.44E+06 7.38E−03 3.03E−09 F5.1.11 2.21E+066.88E−03 3.12E−09 F4.7.8 2.31E+05 3.10E−03 1.34E−08 F5.1.9 2.48E+053.28E−03 1.33E−08 16C3 9.89E+05 6.19E−04 6.37E−10 d1C7 1.13E+06 6.07E−045.36E−10 5344 1.02E+06  <=5.00E−05*     <=5.00E−11     *off-rate wasoutside the specifications of the instrument, an off-rate of 5.0E−05 orless has been assumed

The mIL-2 and hIL-2 binding studies with antibodies F5.1.11, d1C7,13A10, and 16C3 indicate that F5.1.11 and d1C7 do not bind to mIL-2 andthat 13A10 and 16C3 bind mIL-2 with reduced affinity compared to hIL-2(data not shown).

Example 2. IL-2/Antibody Complex Receptor Binding

The ability of the antibodies to modify IL-2 binding to the IL-2receptors IL-2Rα and IL-2Rβ was assessed using a Biacore T200 Instrument(GE Healthcare, Piscataway, N.J.). Recombinant IL-2α and IL-2Rβ proteinwas biotin labeled and captured on a Biacore Streptavidin chip (GEHealthcare). Experiments were performed at 25° C. using a 10 μL/minuteflow rate in 10 mM HEPES (pH 7.4), 150 mM NaCl and 0.005% v/v SurfactantP20 buffer. IL-2 (333 nM) was pre-incubated with IgGs (1000 nM) for atleast 20 minutes. IL-2/antibody complexes were injected over thereceptor coupled chip surface for 60 seconds and allowed to dissociatefor 20 minutes. Data was background subtracted using the adjacentcontrol flow cell and buffer only injections. The response is reportedas the binding to IL-2α and IL-2Rβ after 60 seconds as a percentage ofthe binding of two representative clones, d1C7 and 16C3 in complex withIL-2, to IL-2Rα and IL-2Rβ respectively.

Results

The clone 16C3/IL-2 complex bound IL-2Rβ but not IL-2Rα whereas thed1C7/IL-2 complex bound to IL-2Rα but not IL-2Rβ. These twoantibody/IL-2 complexes were used as representative clones to normalizefor IL-2Rα and IL-2Rβ binding. Each of the tested antibody/IL-2complexes is reported as their relative binding to each of thereceptors. Clone 4.7.062, F5.1.11, F4.7.6, F4.7.8, F5.1.9 and thecommercially available 5344 (Clone 5344.111, BD Biosciences) allinhibited IL-2 from binding to IL-2Rβ similar to the d1C7/IL-2 complexand showed reduced binding to IL-2Rα compared to the d1C7/IL-2 complex.Thus these antibodies were identified for their ability to block IL-2binding to IL-2Rβ and reduce the rate of binding to IL-2Rα (FIG. 1).

Example 3. Antibody/IL-2 Affinity and Receptor Binding for AffinityMatured Clones

The binding kinetics and receptor binding profile of theaffinity-matured F5.1.11 antibody/IL-2 complexes was measured usingsurface plasmon resonance as described for the parental antibody(Example 1).

Results

The affinity maturation campaign increased the apparent binding affinityof clone F5.1.11 for IL-2 up to 16-fold. The three affinity-maturedvariants, F5.1.11.02, F5.1.11.04 and F5.1.11.08, were measured to havebinding affinities for IL-2 of 114, 624 and 205 μM respectively.Characterization of the antibody/IL-2 complex binding to IL-2Rα andIL-2Rβ found that the receptor binding profile of the higher affinityvariants was maintained compared to the parental molecule. Both theparental and affinity matured clones showed complete inhibition of theantibody/IL-2 complex binding to IL-2Rβ and a reduction in the bindingto IL-2Rα compared to the clone d1C7/IL-2 complex. Some variation in theparental F5.1.11/IL-2 binding to IL-2Rα was observed over the course ofthe experiment, this is represented by a dotted line on the graph todepict the potential range. The three higher affinity variants,F5.1.11.02, F5.1.11.04 and F5.1.11.08, all fall within this range (Table4 and FIG. 2).

TABLE 4 clone k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) F5.1.11 3.78E+067.11E−03 1.88E−09 F5.1.11.02 5.05E+06 5.74E−04 1.14E−10 F5.1.11.047.34E+05 4.58E−04 6.24E−10 F5.1.11.08 2.21E+06 4.53E−04 2.05E−10

Example 4. Phenotype of Tregs after IL-2:Anti-IL-2 mAb TreatmentIL-2-Anti-IL-2 Complex Treatment

PBMCs were isolated from healthy donors by ficoll and activatedovernight with 12.5 ng/mL antiCD3 and 25 ng/mL antiCD28. After an overnight incubation, cells were harvested and extensively washed in PBS andresuspended at the concentration of 30×10⁶ cells in 200 μL of PBS. PBMCswere injected i.v. in the NSG recipients. The NSG mice injected withhuman PBMCs received an intraperitoneal injection for 5 days of 8,000 UhIL-2 (Proleukin) complexed with 25 μg of Isotype, 25 μg of 16C3, 1 μg,5 μg or 25 μg of Ab F5.1.11.02 for 30 minutes at 37° C.

Isolation of Splenocytes and Cell Staining

Mice were sacrificed with CO₂ and splenocytes were harvested and stainedextracellularly with anti human CD45 Pacific Orange, CD4 PercPCy 5.5,CD3 PeCy7/Pacific Blue/PercP, CD25 APC-Cy7/Pe, CD238 Pe, CD39 PeCy7, CD8APC-H7, Ki67 Pe, NKG2D PeCy7, CD44 V450 and intracellularly with antihuman Helios FITC and FoxP3 APC. Labeled antibodies were purchased fromBD PharMingen or Ebioscience. Stained single cells suspensions wereanalyzed with a Fortessa flow cytometer running FACSDiva (BDBiosciences) and FSC 3.0 files analyzed and presented with FLOWJOSoftware.

Results

The antibody 16C3 in complex with hIL-2 increased Treg percentage in thespleen. An incremental increase in Treg percentage was also observed inresponse to treatment with two different doses (5 μg and 25 μg) of theantibody F5.1.11.02. Treg population was gated on hCD45⁺ CD3⁺ CD4⁺Helios⁺ FoxP3⁺ cells (FIG. 3). Both 16C3 and F5.1.11.02 in complex withhIL-2 increase Treg/Teff and Treg/NK cell ratios.

16C3 (25 μg) in complex with hIL-2 increased Treg/CD4, Treg/CD8 andTreg/NK cell ratios. The treatment with F5.1.11.02 at the lowest dose (1μg) in the first experiment did not show an increase in the ratio.Instead a significant increase in Treg/CD4, Treg/CD8 and Treg/NK cellratios in response to 5 μg and 25 μg of F5.1.11.02 in complex with hIL-2was observed (FIGS. 4A-C). In the second experiment, the same resultsfor 16C3 and 5 μg and 25 μg of F5.1.11.02 were observed, and, inaddition, an increase in the ratio was also evident with the low dose ofF5.1.11.02 (1 μg) (FIGS. 4D-F). Both 16C3 and F5.1.11.02 in complex withhIL-2 increase Teff and Treg total cell numbers. 16C3 (25 μg) in complexwith hIL-2 increased the total number of CD4⁺, CD8⁺ cells and Tregs inthe spleen in the two experiments.

F5.1.11.02 in complex with hIL-2 also showed an increase in CD4⁺, CD8⁺,and Tregs total cell numbers. The effect was more evident at the dosesof 5 μg and 25 μg of F5.1.11.02 in the first experiment (FIGS. 5A-C).The second experiment showed an increase in total numbers of CD4⁺, CD8⁺cells and Tregs in response to F5.1.11.02 in a dose dependent manner(FIGS. 5D-F). The treatment with F5.1.11.02 also induced an increase ofCD25, Icos and FoxP3 mean fluorescence intensity (MFI) on Tregs that wasnot observed in presence of 16C3 (FIGS. 6A-C).

Example 5. Anti-IL-2 Antibodies Inhibit pSTAT5

Splenocytes from C57B16 expressing GFP under control of the Foxp3promoter were harvested, processed to a single cell suspension, andre-suspended in RPMI 0.1% BSA. Cells were rested at 37° C. in tissueculture incubator until time of assay (1-2 hours).

PBMCs were purified from human Trima residuals (Blood Centers of thePacific) and re-suspended in RPMI 0.1% BSA. Cells were rested at 37° C.in tissue culture incubator until time of assay (1-2 hours).

IL-2-Induced pSTAT5 Assay

PBMCs or splenocytes were plated in a 96 well V-bottom plate, such that1 million cells were in each well, in a volume of 50 μL (RPMI, 0.1%BSA). The plate was returned to 37° C. incubator to maintaintemperature. Antibody (JES6-1 (JES6-1A12 eBioscience) or anti-humanIL-2) was titrated, and IL-2 was tested at 4 concentrations: 500 ng/mL,50 ng/mL, 5 ng/mL, and 0.5 ng/mL.

Commercially available mouse IL-2 monoclonal antibody JES6-1 blocks boththe IL2Rα and IL2Rβ interfaces of IL-2 (León et al. 2013, “Mathematicalmodels of the impact of IL2 modulation therapies on T cell dynamics,”Frontiers in Immunology, 4:439, incorporated in its entirety herein byreference). JES6-1 binds mIL-2 and not hIL-2 because the epitope is notconserved in hIL-2. Key amino acid residues for binding of JES6-1 toIL-2 including mIL-2 residues Q36 and E37, which correspond to hIL-2residues Q22 and M23, are detailed in FIGS. 3, 4, S2, S3, and S4 ofSpangler et al. 2015, “Antibodies to Interleukin-2 Elicit Selective TCell Subset Production through Distinct Conformational Mechanisms,”Immunity, 42: 815, incorporated in its entirety herein by reference. TheKD of mIL-2/mIL-2Rβ is reported by Spangler et al. as >7 μM, and formIL-2/mIL-2Rα the KD is 2-4 fold weaker than in hIL-2. A comparison ofthe mouse and human IL-2 amino acid sequences is available in FIG. 6 ofYokota et al. 1985, “Use of a cDNA expression vector for isolation ofmouse interleukin 2 cDNA clones: Expression of T-cell growth-factoractivity after transfection of monkey cells,” Proc. Natl. Acad. Sci.USA, 82: 68, incorporated in its entirety herein by reference.

Equal volume (50 μL) of 2×IL2:antibody complex (prepared for 1 hr at 37°C.) was added to wells, and cells were cultured in 37° C. incubator for40 minutes. Cells were fixed by addition of 100 μL of IC fix buffer(eBioscience). After 15 minutes fixation, cells were washed and storedin FACs buffer (PBS+0.2% BSA) until staining.

Assay plates were centrifuged to pellet cells, and permeabilizationbuffer Ill (BD Biosciences) was used to re-suspend cells, followed by 30minutes incubation on ice. Cells were washed 2× in FACs buffer, andstained with the following Abs for PBMCs: from eBioscience, anti-humanCD3 APC e780, CD4 Percp e710, and CD127 (PE). CD8 FITC, CD25 (PeCy7),FoxP3 (e660), and pSTAT5 (Pacific blue) were purchased from BDBiosciences. For splenocytes, the following anti-mouse antibodies wereused: from eBioscience CD4 e660 and CD8a PeCy7. pSTAT5 Pacific Blue (BDBiosciences) was used for both mouse and human cells.

Data were collected on LSR Fortessa, and analyzed using FlowJo software.Data are plotted as background subtracted MFI, normalized to max signalfor each cell type (IL2 500 ng/mL+Isotype). Background is defined asnon-stimulated, but stained, pSTAT5. Human Tregs are defined asCD3+CD8-CD4+CD25hiCD127lo. Mouse Tregs are defined as CD8⁻CD4⁺Foxp3.GFP⁺ cells.

Results

Inhibition of pSTAT5 in PBMCs: F5.1.11 Affinity Variants

Affinity variants of F5.1.11 demonstrated that antibodies with increasedaffinity for IL-2, such as F5.1.11.02 and F5.1.11.08, were moreeffective at inhibiting pSTAT5 signaling in CD8⁺ effector T cells, andnon-Treg CD4+ T cells. Treg pSTAT5 was maintained greater than 50% atIL-2 concentrations of 5-500 ng/mL. At the lowest concentration of IL-2tested (0.5 ng/mL) pSTAT5 levels in Tregs were inhibited below 50% ofmax signal, however pSTAT5 was still detectable across all Abconcentrations (FIGS. 7A-V). The 5344 antibody was also tested in thepSTAT5 signaling assay, and in Tregs pSTAT5 was no longer detectable atlow concentrations of IL-2 (data not shown).

Comparison of JES6-1 and Antibody F5.1.11.02

Mouse IL-2 antibody JES6-1 spared Treg pSTAT5 signaling at most IL-2concentrations tested, similar to isotype control (FIGS. 8A-F). Incontrast, IL-2 induced pSTAT5 signaling was strongly inhibited by JES6-1in mouse CD8⁺ T cells, at all IL-2 concentrations tested. This dataagrees with published observations that JES6-1 in complex with IL-2promotes the growth of Treg cells in vivo.

Anti-human IL-2 antibody F5.1.11.02 largely spared human Treg pSTAT5signaling at most IL-2 concentrations tested. CD8⁺ T cell pSTAT5 wasinhibited by IL-2 antibody F5.1.11.02 at all IL-2 concentrations tested.Overall, the pattern of pSTAT5 inhibition of IL-2 antibody F5.1.11.02 inCD8 and Tregs is similar to that observed with mouse IL-2 antibodyJES6-1.

In Table 5, pSTAT5% max values from FIGS. 8A-F were used to generatearea under the curve values for either Tregs or CD8 cells, treated witheither Isotype or anti-IL-2 antibody. The ratio of Treg/CD8 AUC for eachconcentration of IL-2 was listed. In isotype treated samples, decreasingamounts of IL-2 resulted in an increased AUC ratio, reflecting morepSTAT5 signaling in the Tregs than in the CD8⁺ effector cells. Althoughthe absolute numbers differ, this observation was consistent betweenmouse and human cells.

JES6-1 treatment shifted the ratio to be in favor of Tregs at higherconcentrations of IL-2. A similar change in AUC ratio occurred in thehuman cells treated with F5.1.11.02 antibody.

Overall, F5.1.11.02 appeared to have a similar pSTAT5 signaling profileto JES6-1.

Impact of Antibody Epitope on pSTAT5 Inhibition of CD25hi CD8⁺ T Cells

Some peripheral CD8⁺ T cells express CD25 (IL-2Rα). As shown in FIGS.10A-B, if cells were gated based on CD25 expression, CD25hi CD8 cellswere more sensitive to low levels of IL-2. IL-2 antibodies' blocking ofCD25 binding, all or in part, may therefore be required to inhibitpSTAT5 signaling in CD25hi CD8⁺ T cells.

As shown in FIG. 9, pSTAT5 induced by low doses of IL-2 (0.8 ng/mL) wasbest inhibited by IL-2 antibodies that blocked IL-2Rα binding to IL-2,such as 13A10 or F5.1.11.02. Conversely, high concentrations of IL-2(500 ng/mL), presumably not requiring the presence of IL-2Rα to enablesignaling, were best inhibited by antibodies that block binding of IL-2to IL-2Rβ, such as dl C7 or F5.1.11.02. Antibody F5.1.11.02 was bestable to inhibit pSTAT5 signaling in CD8⁺ effector T cells across a rangeof IL-2 concentrations

pSTAT5 Inhibition by IL-2 Antibody F5.1.9

IL-2 antibody F5.1.9 and variant F5.1.9.5 were assessed for inhibitionof pSTAT5 (FIGS. 11A-H). Affinity variant F5.1.9.5 of F5.1.9 waseffective at inhibiting pSTAT5 signaling in CD8⁺ effector T cells to agreater extent than it inhibited pSTAT5 signaling in Tregs. F5.1.9.5k_(a) (M⁻¹s⁻¹) was 1.23E+06, k_(d) (s⁻¹) was 8.18E-04, and K_(D) (M)6.63E-10.

Example 6. Generation of NOD mIL-2−/−hIL-2+/−Mice

A mouse IL-2-containing bacterial artificial chromosome (BAC) (cloneRP23-290D8) was engineered to express human IL-2 coding sequence. Thefollowing modifications were made in the BAC: the mouse IL-2 promoter,5′ and 3′ UTRs and introns were left intact; the mouse signal peptidewas replaced with the human one; the 3′ half of exon 1 downstream ofsignal peptide, exons 2 and 3 were replaced with human sequence; the 5′half of exon 4 was replaced with human coding sequence.

The engineered BAC was subsequently transferred through a pronuclearinjection in fertilized oocytes of an FVB/NJ (Friend Virus B) femalemouse (Jackson).

The fertilized oocytes were then implanted in an FVB female mouse togenerate FVB mIL-2+/+hIL-2 BAC+mice. In order to generate mice in NODbackground who were lacking the expression of mIL2, the FVBmIL-2+/+hIL-2 BAC+mice were backcrossed with a NOD mIL-2−/−mouse. TheNOD mIL-2−/−hIL-2 BAC+/−mice generated from this initial breeding, werebackcrossed with a NOD mIL-2−/−mouse for 8 generations in order togenerate mice with type I diabetes. The mice obtained were used forexperiments.

Methods

Whole blood (100 μL), after ACK lysis, was stimulated overnight withPMA/lonomycin, the supernatant (125 μL) was collected and the ELISAperformed for both the mIL-2 (50 μL) and hIL-2 (50 μL) (ELISA:ebioscience).

The female NOD mIL2−/−hIL2+/−mice were injected at the age of 14 weeksand they received an intraperitoneal injection for 5 days of 8,0001 UhIL-2 (Proleukin) in complex with 25 μg of isotype, or 5 μg or 25 μg ofF5.1.11.02 antibody. IL2:antibody complexes were formed by incubatingIL-2 and antibody together for 30 minutes at 37° C.

Isolation of Different Organs and Cell Staining

Mice were sacrificed with CO₂ and immediately perfused through the leftventricle with PBS until the effluent ran clear. Splenocytes andpancreatic lymph nodes (pLN) were harvested and extracellular stainingwith CD25 PeCy7, CD4 BV605, CD8 PercP-Cy5.5, CD44 Pe, CD62L A1647 andintracellular staining with FoxP3 FITC was performed.

Whole pancreas was prepared by digesting for 30 minutes at 37° C. with0.8 mg/mL Collagenase P and 20 μg/mL DNase (Roche) followed by mincing.After digestion, the homogenate was filtered twice by using a 40 μm cellstrainer and extra-cellular staining with CD45 PercP Cy5.5, CD25 Pe, CD4PeCy7, CD8 Al647, Thy1.2 BV605, and intracellular staining with FoxP3FITC, was performed. Labeled antibodies were purchased from BDPharmingen or Ebioscience. Stained single cell suspensions were analyzedand presented with FLOWJO software.

Results

Phenotype of Human IL-2 Transgenic (hIL-2 Tg) Mice

In vitro stimulation of mouse splenocytes (FIGS. 12A-B) withPMA/lonomycin confirmed the production of human IL-2 from transgenicmice, although human IL-2 was produced at lower levels than mouse IL2from wildtype mice. hIL-2 Tg I/mIL-2−/−mice appeared healthy and viable.However, hIL-2Tg mice had an increased percentage of activated(CD44+CD62L−) CD4⁺ or CD8⁺ T cells, as compared to NOD or NODmIL-2+/−mice (FIGS. 13A-B). Although Treg frequency was similar to NODor NOD mIL-2+/−mice, Tregs from hIL-2 Tg mice had a decreased cellsurface expression of CD25 (FIG. 14).

Treatment of hIL-2 Tg Mice with F5.1.11.02:hIL-2 Complex

hIL-2 Tg mice were treated with F5.1.11.02:IL-2 complex, as describedabove (methods). Treatment did not increase overall cellularity of thespleen at day 7, and a slight decrease in total splenocytes was observedin the 5 μg treatment group (FIG. 15). An increase in Treg percentagewas observed in response to treatment with both doses of F5.1.11.02 incomplex with hIL-2 compared to the isotype control in all organs exceptpLNs, with a more significant increase in the pancreas (FIGS. 16A-I). Inthe spleen and pLNs, the Treg population was gated on CD4⁺ CD25⁺ FoxP3cells and in the pancreas Treg cells were identified by gating on CD45′Thy1.2⁺ CD4⁺ CD25⁺ FoxP3⁺ cells.

After treatment with the complex, a decrease of CD4, CD8 and Treg totalcell numbers was observed in the spleen and pLN. However, in thepancreas, F5.1.11.02 complex treatment induced a slight increase in Tregtotal cell number in response to the 25 μg dose of F5.1.11.02. Nodifferences in CD4 and CD8 total cell numbers were observed in thepancreas (data not shown).

Treatment of hIL-2 Tg mice with F5.1.11.02 (25 μg) in complex with hIL-2induced a significant increase in Treg/CD4 and Treg/CD8 cell ratios inthe spleen (FIGS. 17A-F). The same results were observed in thepancreas, with a significant increase in ratio for both Treg/CD4 andTreg/CD8 observed with 5 μg and 25 μg of F5.1.11.02. F5.1.11.02:1L2complex treatment had little effect on cell ratios in the pancreaticlymph node.

Lastly, treatment of huIL2 Tg mice with F5.1.11.02:IL-2 complex (5 μgand 25 μg F5.1.11.02) induced an increase of CD25 mean fluorescenceintensity (MFI) on Tregs in all organs (FIGS. 18A-C). This increase wasparticularly significant in the pancreas, where Treg CD25 MFI was verylow prior to treatment.

NSG Experiment with CTV

PBMCs were isolated from healthy donors by ficoll and activatedovernight with 12.5 ng/mL antiCD3 and 25 ng/mL antiCD28. After an overnight activation, cells were harvested and labeled with CellTrace Violet(Life technology) at the concentration of 1 μL CTV/10×10⁶ cells. Afterthe staining, the cells were extensively washed in PBS and resuspendedat the concentration of 30×10⁶ cells in 200 μL of PBS. PBMCs wereinjected i.v. in the NSG recipients. The NSG mice injected with humanPBMCs received an intraperitoneal injection daily for 5 days of 8,000 UhIL-2 (Proleukin) in complex with isotype or antibody, in the amountsindicated, for 30 minutes at 37° C.

Isolation of Splenocytes and Cell Staining

Mice were sacrificed with CO₂ at day 3 and day 5 and splenocytes wereharvested and stained extracellularly with anti human CD45 PacificOrange, CD4 PercPCy5.5, CD25Pe, CD8 APC-H7, NKG2D PeCy7 andintracellularly with anti human Helios FITC and FoxP3 APC. Labeledantibodies were purchased from BD PharMingen or Ebioscience. Stainedsingle cells suspensions were analyzed with a Fortessa flow cytometerrunning FACSDiva (BD Biosciences) and FSC 3.0 files analyzed andpresented with FLOWJO Software.

Results

The treatment with 25 μg of F5.1.11.02 in complex with hIL-2 induced anincrease in the number of total splenocytes at day 5 but not at day 3(FIGS. 19A-B).

F5.1.11.02 antibody:IL-2 complex was also able to increase Teff and Tregtotal cell number (FIGS. 20A-F) and Treg/CD4 and Treg/CD8 ratios (FIGS.21A-D) in the spleen at day 5, a result that was not observed at day 3.Treg population was gated on hCD45⁺ CD3⁺ CD4⁺ Helios⁺ FoxP3⁺ cells.

By including CTV labeling prior to transfer and treatment, we observedthat treatment with F5.1.11.02 antibody:IL-2 complex inducedproliferation of Tregs compared to isotype, an effect already evident atday 3 and resulting in a low number of undivided cells by day 5 (FIGS.22A-H). Proliferation of CD8 T cells was also increased by treatmentwith F5.1.11.02 antibody:IL-2 complex, however this was only significantat day 5 and greater numbers of undivided cells remained than in theTreg population (FIGS. 23A-H). Overall, this CTV data supports anincrease in Treg proliferation as underlying the shift in Treg/CD8 ratioobserved.

Lastly, in the spleen the treatment with both doses of the complexinduced an increase of CD25 mean fluorescence intensity (MFI) on Tregsand on the CD8 population compared to the isotype control (FIGS. 24A-B).However, the absolute expression of CD25 on Tregs was many fold higherthan on CD8 cells.

In a follow-on experiment, we compared F5.1.11.02 with a lower affinity,‘parental’ antibody, F5.1.11. We extended the dose range to 125 μg ofantibody, in complex with hIL2, to assess if a higher dose of a loweraffinity antibody would have a comparable effect in vivo. At 125 μg,F5.1.11 had comparable effects on Treg and CD8 cell number, increasedTreg/CD8 ratio, and cellularity, equivalent to 25 μg of F5.1.11.02(FIGS. 25A-D, 26A-C, and 33A-B). This was again supported by increasedTreg proliferation, as observed in the CTV dilution (FIGS. 27A-H, 28A-H,and 38A-B). We predict that in vitro, F5.1.11.02 will similarly increaseFoxp3 and CD25 protein levels on human Tregs.

Example 7. IL-2 Antibody Binding Affinity for Cynomolgus Monkey IL-2

Recombinant cynomolgus monkey (Macaca fascicularis) IL-2 was expressedin mammalian cells and purified using immobilized metal ion affinitychromatography (IMAC). The affinity of the antibodies for cynomolgusmonkey IL-2 (1-100 nM) was measured using surface plasmon resonance asdescribed for the human IL-2 (Example 1). A 1:1 Langmuir binding modelwas used to fit binding curves or the steady-state affinity constantcalculated from a concentration-response curve. Clones were designatedas “very weak” in cases where the concentration-response relationshipobserved for 1-100 nM IL-2 was insufficient to calculate the apparentbinding affinity.

Results

The human antibody clones tested bind to cynomolgus monkey IL-2 withaffinities ranging from 516 μM through to very weak associations thatcould not be quantified in this assay (Table 6). The majority of clonestested where selected based on their ability to inhibit IL-2Rβ binding(Examples 2 and 3), these clones showed a significant loss of affinityfor the cynomolgus monkey IL-2 when compared to human IL-2. 16C3, whichhas been observed to inhibit IL-2Rα binding (Example 2), showedequivalent binding to both human and cynomolgus monkey IL-2. Theseresults are consistent with our structural analysis which found thedivergent region between human and cynomolgus monkey IL-2 to fall withinboth the IL-2Rβ binding site and the epitope of the F5.1.11 antibodyclone (Example 9).

TABLE 6 Clone Human IL-2 KD (M) Cyno IL-2 KD (M) F5.1.11 3.12E−09 veryweak F4.7.8 1.34E−08 9.24E−08 F5.1.9 1.33E−08 very weak 16C3 6.37E−105.16E−10 d1C7 5.36E−10 >=1.08E−07     5344  <=5.00E−11*     >=5.0E−08     F5.1.11.02 1.14E−10 ~2.11E−07   *off-rate was outside thespecifications of the instrument, an off-rate of 5.0E−05 or less hasbeen assumed.

Example 8. IL-2/Fab Complex Receptor Binding Affinity

The affinity of IL-2Rα for IL-2 or the IL-2/F5.1.11 Fab complex wasmeasured using a Biacore T200 Instrument (GE Healthcare, Piscataway,N.J.). Recombinant IL-2a was biotin labeled and captured on a BiacoreStreptavidin chip (GE Healthcare). Experiments were performed at 25° C.using a 10 μL/minute flow rate in 10 mM HEPES (pH7.4), 150 mM NaCl and0.005% v/v Surfactant P20 buffer. IL-2 (1-50 nM) or IL-2/F5.1.11 Fab(1-500 nM IL-2 with excess Fab pre-complexed) was were injected over thereceptor coupled chip surface for 120 seconds at 30 μL/minute andallowed to dissociate for 5 minutes. Data was background subtractedusing the adjacent control flow cell and buffer only injections. Theaffinity of the interaction was calculated from concentration-responserelationship at equilibrium.

Results

The IL-2/F5.1.11 Fab complex binds to IL-2Rα with a lower affinitycompared to unliganded IL-2. Equilibrium binding analysis of the complexand unliganded IL-2 found the F5.1.11 Fab bound IL-2 has anapproximately 7-fold weaker affinity for IL-2Rα (9.97 nM compared to70.1 nM) (FIGS. 29A-B). This result is consistent with the reducedbinding observed in the IL-2/antibody complex receptor binding assay(Example 2) and the analysis of the IL-2/F5.1.11 Fab crystal structure(Example 9).

Example 9. Crystal Structure of the F5.1.11 Fab Bound to Human IL-2Interleukin-2 Expression and Purification

Full-length IL-2 (residues 1-133) was purified as described (Rickert etal., 2004). Briefly, the gene was cloned into the pAcgp67A vectorin-frame with the gp67A signal sequence of the vector and followed by aC-terminal hexahistidine tag (SEQ ID NO: 223). Spodoptera frugiperda(Sf9) cells were used to generate high-titer recombinant virus.Trichopulsia ni (High-Five) cells grown in Insect Xpress medium (Lonza)were infected with the virus and allowed to express protein for 48 hoursat 28° C. The protein was purified by Ni-NTA and digested overnight withcarboxypeptidases A and B at 4° C., then purified on a Superdex 200 gelfiltration column (GE Healthcare).

Crystallization of 5.1.11 Fab Complex with Interleukin-2

F5.1.11 Fab was mixed with a 1.5-fold excess of IL-2 and the complex waspurified by FPLC on a Superdex 200 column (GE Healthcare). The purifiedFab/IL-2 complex was concentrated to 10 mg/mL as measured at 280 nmusing an extinction coefficient of 1.49 mL*mg⁻¹*cm⁻¹. The complex wasmixed with an equal volume of precipitant solution (100 mM sodiumcitrate, pH 5.0; 20% polyethylene glycol 6000) and crystallized bysitting drop vapor diffusion over a reservoir of precipitant solution.Prismatic crystals formed within one week. Crystals were cryoprotectedby addition of glycerol to 30% (v/v), harvested, and rapidly cooled byplunging into liquid nitrogen.

Crystallographic Data Collection and Refinement

Diffraction data were collected at the Advanced Light Source Beamline8.2.1 (Berkeley, Calif.). The crystals were indexed in space group P2₁with unit cell dimensions a=86.01 Å, b=145.73 Å, c=107.32 Å, β=95.38°. Amaximum resolution of 2.75 Å was used for structure solution andrefinement. The structure was solved by molecular replacement using theprogram Phaser (McCoy et al., 2007). Search models included the heavychain constant domain from PDB entry 3U1 S, the heavy chain variabledomain from 4NPY, the light chain constant domain from 3N9G, the lightchain variable domain from 4HPO, and interleukin-2 from 2B5l (Wang etal., 2005; McLellan et al., 2011; Ogata et al., 2013; Sok et al., 2013;Kaufmann et al.). Four copies of the Fab/IL-2 complex were modeled inthe asymmetric unit. The structure was built by iterative cycles ofmanual rebuilding and refinement using the programs Coot (Emsley et al.,2010) and PHENIX (Adams et al., 2010). Protein-protein interactions wereanalyzed by visual inspection in Coot and with PISA (Krissinel andHenrick, 2005; 2007). A homology model of cynomologus monkey (Macacafascicularis) IL-2 was prepared with I-TASSER (Roy et al., 2010; Yang etal., 2015), using the human apo-IL-2 (PDB ID 1M47 (Arkin et al., 2003))as a template.

Results

F5.1.11 interacts with the I L-2 at helices A and C and the B-C loop viathe light chain CDR1 and CDR3 loops and the heavy chain CDR2 and CDR3loops (FIG. 30). Comparison to the IL-2/IL-2Rβ/γ_(c)/CD25 quaternarystructure (PDB ID 2B5l) reveals that F5.1.11 occludes the IL-2Rβ bindingsite of IL-2 but not the CD25 or γ_(c) binding sites. The overallstructure of IL-2 is similar to the apo structure (RMSD 0.359 Å over 95Cα atoms). A significant perturbation occurs where F5.1.11 binds the B-Cloop of IL-2; this perturbation is propagated to the adjacent IL-2 A-Bloop (FIG. 31). Since the A-B loop forms part of the CD25 binding sitein the quaternary complex, this movement may explain the decreasedaffinity of F5.1.11/IL-2 for CD25.

To explain the weak cross-reactivity of F5.1.11 to cynomolgus monkeyIL-2, we generated a homology model of cynomolgus monkey IL-2 based onthe apo human IL-2 structure. Although the sequences are 95% identical,an insertion in the B-C loop of cyno IL-2 appears likely to disrupt theinteraction with the light chain CDR1 residues (Ala30, Ser31, Asn32, andTyr33) and heavy chain CDR3 residues (Gly105 and Asp106) (FIG. 32).

In order to improve binding of mAb F5.1.11.02 to non-human primate IL-2,target mAb residues whose side chains contact the IL-2 loop that variesbetween human and cyno IL-2 were chosen based on the F5.1.11 Fab/hIL-2crystal structure. Codons corresponding to residues Gly105, Asp106 ofCDRH3 in the heavy chain and Ala30, Ser31, Asn32, Tyr33 of CDRL1 in thelight chain were randomized combinatorially by PCR with degenerateoligonucleotides encoding 19 amino acids (excluding cysteine). Theresulting library in scFv format was screened by surface display methodsfor binding to both cyno and human IL-2.

Example 10. Diabetes Remission by IL-2 Complex Anti-IL-2:IL-2 ComplexTreatment

PBMCs were isolated from healthy donors by ficoll and activatedovernight with 12.5 ng/mL antiCD3 and 25 ng/mL antiCD28. After an overnight incubation, cells were harvested and labeled with CellTrace Violet(Life technology) at the concentration of 1 μL CTV/10×10⁶ cells. Afterthe staining, the cells were extensively washed in PBS and resuspendedat the concentration of 30×10⁶ cells in 200 μL of PBS. PBMCs wereinjected i.v. in NSG recipients. Antibody:IL-2 complexes were formed byincubating isotype or anti-IL-2 antibody, at concentrations indicated,with 8000 U human IL2 (Proleukin) for 30 minutes at 37° C. The NSG miceinjected with human PBMCs received an intraperitoneal injection dailyfor 5 days of antibody:IL-2 complex.

Isolation of Splenocytes and Cell Staining

Mice were sacrificed with CO₂ and splenocytes were harvested and stainedextracellularly with anti human CD45 Pacific Orange, CD4 PercPCy 5.5,CD3 PeCy7/Pacific Blue/PercP, CD25 APC-Cy7/Pe, CD238 Pe, CD39 PeCy7, CD8APC-H7, Ki67 Pe, NKG2D PeCy7, CD44 V450 and intracellularly with antihuman Helios FITC and FoxP3 APC. FoxP3 buffer kit (eBioscience) was usedfor fixation and permeabilization, as per manufacturer's instructions,prior to intracellular staining. Labeled antibodies were purchased fromBD PharMingen or Ebioscience. Stained single cells suspensions wereanalyzed with a Fortessa flow cytometer running FACSDiva (BDBiosciences) and FSC 3.0 files analyzed and presented with FLOWJOSoftware.

Diabetes Remission by IL2 Complex

Spontaneous new-onset diabetic NOD mice (one blood glucose concentrationbetween 250 mg/dl and 350 mg/dl) were treated for 5 days with 8,000 UhIL-2 (Proleukin) complexed with 125 μg of Isotype or 125 μg ofF5.1.11.02 and 0.5 μg mIL2 (Ebioscience) complexed with 5 μg of JES6-1(Ebioscience). Blood glucose concentrations were monitored over time.

After 1 week of remission, some mice were sacrificed and the pancreaswas analyzed. Whole pancreas was prepared by digesting for 30 minutes at37° C. with 0.8 mg/mL Collagenase P and 20 μg/mL DNase (Roche) followedby mincing. After digestion, the homogenate was filtered twice by usinga 40 μm cell strainer and stained extracellularly with CD45 PercP Cy5.5,CD25 Pe, CD4 PeCy7, CD8 Al647, Thy1.2 BV605 and intracellularly withFoxP3 FITC. Labeled antibodies were purchased from BD Pharmingen orEbioscience. Stained single cell suspensions were analyzed and presentedwith FLOWJO software.

Results

The treatment with F5.1.11.02 in complex with hIL2 (F5.1.11.02:IL2complex) induced an increase in the percent of Tregs in the spleen. Tregpopulation was gated on hCD45⁺ CD3⁺ CD4⁺ Helios⁺ FoxP3⁺ cells.F5.1.11.02 antibody:IL-2 complex increased Tregs total cell number,although an increase in total CD4 and CD8 cell number was also observed(FIGS. 35 A-B). Overall, the ratios of Treg/CD4 and Treg/CD8 (FIG. 36A)were increased in a dose dependent manner. The effect was significantstarting at the dose of 1 μg.

By including CTV labeling prior to transfer and treatment, we observedthat treatment with F5.1.11.02 antibody:IL-2 complex induced increasedproliferation of Tregs compared to isotype. Proliferation of CD8 T cellswas also increased by treatment with F5.1.11.02 antibody:IL-2 complex,however greater numbers of undivided cells remained in the CD8population than in Treg population (FIG. 36B). Overall, this CTV datasupports an increase in Treg proliferation as underlying the shift inTreg/CD8 ratio observed.

In the spleen the treatment with all doses of the complex induced anincrease of CD25 mean fluorescent intensity (MFI) on Tregs and CD8population compared to isotype control. However, the absolute expressionof CD25 on Tregs was many folds higher than on CD8 cells (FIGS. 37A-B).In addition, after treatment with F5.1.11.02:IL-2 complex, an increasein FoxP3 MFI on Tregs was also observed (FIG. 38). Together, these datasuggest that the expanded Tregs maintain phenotype and function.

In vitro data have suggested that anti-1L2 antibodies that block thebinding of IL2Rb to IL-2 will be better able to promote differentialTreg expansion. In order to directly test this hypothesis, we comparedantibodies that blocked different receptor epitopes on IL-2: an IL-2Rαblocker (16C3.4), an IL2Rβ blocker (d1C7), and an IL2Rβ blocker thatalso reduced IL2's binding to IL-2Rα (F5.1.11.02). All three antibodiesin complex with hIL2 increased Treg cell number, and to a lesser extentCD4 and CD8 cell numbers also increased. The increase in CD8 cell numberwas much greater in response to 16C3.4 than to either of the IL-2Rbblocking antibodies (d1C7 or F5.1.11.02) (FIG. 39A).

Consistent with the altered cell numbers, d1C7 and F5.1.1.11.02:IL-2complex showed an increase in Treg/CD4 and Treg/CD8 ratios, which wasnot observed with 16C3.4 (FIG. 39B). The treatment with d1C7 andF5.1.11.02 in complex increased CD25 MFI on Treg and CD8, however theCD25 expression on CD8 was higher with d1C7, suggesting that F5.1.11.02might be more selective for Tregs (FIG. 40).

Lastly, we assessed if 5 days of F5.1.11.02:IL-2 complex administrationcould be effective to cure clinical diabetes in NOD mice. Remarkably,this treatment induced diabetes remission in 50% of the mice within 1week and most of them remained normoglycemic over the 4 week duration ofthe experiment. JES6-1:mouse IL-2 complex provided a less curativeeffect than F5.1.11.02 and no effect was observed in response to isotype(FIG. 41).

To establish whether the F5.1.11.02 antibody:IL-2 complex effect wasrelated to a modification of Treg numbers and characteristics in thepancreas, we quantified their proportion in the pancreas after 1 week ofremission. We observed that the percentage of pancreatic Treg wassignificantly increased after treatment with F5.1.11.02 in complex. Wealso observed that the treatment with the complex was associated with arise in the expression of markers associated with Treg cell function,such as CD25 and FoxP3, which was not observed with isotype control(FIG. 42). Overall these data suggest that Treg increase in the pancreasafter treatment might control the progression to destructive insulitisin diabetic mice.

REFERENCES

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Detection of protein assemblies in crystals.    Berthold M R, Glen R C, Diederichs K, Kohlbacher O, Fischer I,    editors. Computational Life Sciences. Berlin: Springer; 2005. p.    163-74.-   Krissinel E, Henrick K. Inference of Macromolecular Assemblies from    Crystalline State. J Mol Biol. 2007 September; 372(3):774-97.-   McCoy A J, Grosse-Kunstleve R W, Adams P D, Winn M D, Storoni L C,    Read R J. research papers. J. Appl. Cryst (2007). 40, 658-674 [doi:    10.1107/S0021889807021206]. International Union of Crystallography;    2007 Jul. 13; 1-17.-   McLellan J S, Pancera M, Carrico C, Gorman J, Julien J-P, Khayat R,    et al.-   Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing    antibody PG9. Nature. Nature Publishing Group; 2011 Dec. 15;    480(7377):336-43.-   Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, et al. A    Novel Transition-state Analogue for Lysozyme,    4-O-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence    Supporting the Covalent Glycosyl-enzyme Intermediate. Journal of    Biological Chemistry. 2013 Mar. 1; 288(9):6072-82.-   Rickert M, Boulanger M J, Goriatcheva N, Garcia K C. Compensatory    energetic mechanisms mediating the assembly of signaling complexes    between interleukin-2 and its alpha, beta, and gamma(c) receptors. J    Mol Biol. 2004 Jun. 18; 339(5):1115-28.-   Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for    automated protein structure and function prediction. Nat Protoc.    2010 April; 5(4):725-38. PMCID: PMC2849174-   Sok D, Laserson U, Laserson J, Liu Y, Vigneault F, Julien J-P, et    al. The Effects of Somatic Hypermutation on Neutralization and    Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies.    PLOS Pathog. Public Library of Science; 2013 Nov. 21;    9(11):e1003754.-   Wang X, Rickert M, Garcia K C. Structure of the quaternary complex    of interleukin-2 with its alpha, beta, and gammac receptors.    Science. 2005 Nov. 18; 310(5751):1159-63.-   Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite:    protein structure and function prediction. Nat Methods. 2015;    12:7-8.    Particular embodiments of the invention are set forth in the    following numbered paragraphs:    1. An isolated antibody or an antigen-binding portion thereof that    specifically binds human IL-2 (hIL-2), wherein the antibody binds    helices A and C and the B-C loop of hIL-2.    2. An isolated antibody or an antigen-binding portion thereof that    competes for binding to human IL-2 (hIL-2) with, or binds the same    epitope of hIL-2 as, an antibody comprising the amino acid sequences    of SEQ ID NOs: 13 and 14.    3. An isolated antibody or an antigen-binding portion thereof that    specifically binds human IL-2 (hIL-2), wherein the antibody reduces    the binding affinity of hIL-2 to IL-2Rα by 1 to 199 fold.    4. The antibody or antigen-binding portion of paragraph 3, wherein    the antibody reduces the binding affinity of hIL-2 to IL-2Rα by 10    fold.    5. An isolated antibody or an antigen-binding portion thereof that    specifically binds human IL-2 (hIL-2), wherein the antibody reduces    hIL-2 binding to IL-2Rα and IL-2Rβ, and inhibits an activity in CD8⁺    T cells to a higher degree than in regulatory T (Treg) cells.    6. An isolated antibody or an antigen-binding portion thereof that    specifically binds human interleukin-2 (hIL-2), wherein the antibody    reduces hIL-2 binding to IL-2Rα and IL-2Rβ, and inhibits STAT5    phosphorylation in CD8⁺ T cells to a higher degree than in    regulatory T (Treg) cells.    7. An isolated antibody or an antigen-binding portion thereof that    specifically binds human interleukin-2 (hIL-2), wherein the antibody    reduces hIL-2 binding to IL-2Rα and IL-2Rβ, and increases the ratio    of regulatory T (Treg) cells to CD8⁺ or CD4⁺ T cells or to NK cells    as measured in a peripheral blood mononuclear cell (PBMC) culture or    reconstitution assay.    8. An isolated antibody or an antigen-binding portion thereof that    specifically binds human interleukin-2 (hIL-2), wherein the antibody    reduces hIL-2 binding to IL-2Rα and IL-2Rβ, and increases expression    of one or more of FOXP3, CD25, and Icos in regulatory T (Treg)    cells.    9. The isolated antibody or antigen-binding portion of any one of    paragraphs 1-8, wherein the antibody or antigen-binding portion has    at least one of the following properties:

a) binding to hIL-2 with an off-rate greater than about 4.53×10⁻⁴ s⁻¹;and

b) binding to hIL-2 with a K_(D) greater than about 1.14×10⁻¹⁰ M.

10. The antibody or antigen-binding portion of any one of paragraphs1-9, wherein the antibody is a human antibody.11. The antibody or antigen-binding portion of any one of paragraphs1-10, comprising:

(a) a HCDR1 comprising SEQ ID NO: 73 (Kabat), 74 (Chothia), or 75(extended); a HCDR2 comprising SEQ ID NO: 76 (Kabat), or 77 (Chothia); aHCDR3 comprising SEQ ID NO: 78; a LCDR1 comprising SEQ ID NO: 79; aLCDR2 comprising SEQ ID NO: 80; and a LCDR3 comprising SEQ ID NO: 81;

(b) a HCDR1 comprising SEQ ID NO: 82 (Kabat), 83 (Chothia), or 84(extended); a HCDR2 comprising SEQ ID NO: 85 (Kabat), or 86 (Chothia); aHCDR3 comprising SEQ ID NO: 87; a LCDR1 comprising SEQ ID NO: 88; aLCDR2 comprising SEQ ID NO: 89; and a LCDR3 comprising SEQ ID NO: 90;

(c) a HCDR1 comprising SEQ ID NO: 91 (Kabat), 92 (Chothia), or 93(extended); a HCDR2 comprising SEQ ID NO: 94 (Kabat), or 95 (Chothia); aHCDR3 comprising SEQ ID NO: 96; a LCDR1 comprising SEQ ID NO: 97; aLCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;

(d) a HCDR1 comprising SEQ ID NO: 100 (Kabat), 101 (Chothia), or 102(extended); a HCDR2 comprising SEQ ID NO: 103 (Kabat), or 104 (Chothia);a HCDR3 comprising SEQ ID NO: 105; a LCDR1 comprising SEQ ID NO: 106; aLCDR2 comprising SEQ ID NO: 107; and a LCDR3 comprising SEQ ID NO: 108;

(e) a HCDR1 comprising SEQ ID NO: 109 (Kabat), 110 (Chothia), or 111(extended); a HCDR2 comprising SEQ ID NO: 112 (Kabat), or 113 (Chothia);a HCDR3 comprising SEQ ID NO: 114; a LCDR1 comprising SEQ ID NO: 115; aLCDR2 comprising SEQ ID NO: 116; and a LCDR3 comprising SEQ ID NO: 117;

(f) a HCDR1 comprising SEQ ID NO: 118 (Kabat), 119 (Chothia), or 120(extended); a HCDR2 comprising SEQ ID NO: 121 (Kabat), or 122 (Chothia);a HCDR3 comprising SEQ ID NO: 123; a LCDR1 comprising SEQ ID NO: 124; aLCDR2 comprising SEQ ID NO: 125; and a LCDR3 comprising SEQ ID NO: 126;

(g) a HCDR1 comprising SEQ ID NO: 127 (Kabat), 128 (Chothia), or 129(extended); a HCDR2 comprising SEQ ID NO: 130 (Kabat), or 131 (Chothia);a HCDR3 comprising SEQ ID NO: 132; a LCDR1 comprising SEQ ID NO: 133; aLCDR2 comprising SEQ ID NO: 134; and a LCDR3 comprising SEQ ID NO: 135;

(h) a HCDR1 comprising SEQ ID NO: 136 (Kabat), 137 (Chothia), or 138(extended); a HCDR2 comprising SEQ ID NO: 139 (Kabat), or 140 (Chothia);a HCDR3 comprising SEQ ID NO: 141; a LCDR1 comprising SEQ ID NO: 142; aLCDR2 comprising SEQ ID NO: 143; and a LCDR3 comprising SEQ ID NO: 144;

(i) a HCDR1 comprising SEQ ID NO: 145 (Kabat), 146 (Chothia), or147(extended); a HCDR2 comprising SEQ ID NO: 148 (Kabat), or 149(Chothia); a HCDR3 comprising SEQ ID NO: 150; a LCDR1 comprising SEQ IDNO: 151; a LCDR2 comprising SEQ ID NO: 152; and a LCDR3 comprising SEQID NO: 153;

(j) a HCDR1 comprising SEQ ID NO: 154 (Kabat), 155 (Chothia), or 156(extended); a HCDR2 comprising SEQ ID NO: 157 (Kabat), or 158 (Chothia);a HCDR3 comprising SEQ ID NO: 159; a LCDR1 comprising SEQ ID NO: 160; aLCDR2 comprising SEQ ID NO: 161; and a LCDR3 comprising SEQ ID NO: 162;

(k) a HCDR1 comprising SEQ ID NO: 163 (Kabat), 164 (Chothia), or 165(extended); a HCDR2 comprising SEQ ID NO: 166 (Kabat), or 167 (Chothia);a HCDR3 comprising SEQ ID NO: 168; a LCDR1 comprising SEQ ID NO: 169; aLCDR2 comprising SEQ ID NO: 170; and a LCDR3 comprising SEQ ID NO: 171;

(l) a HCDR1 comprising SEQ ID NO: 172 (Kabat), 173 (Chothia), or 174(extended); a HCDR2 comprising SEQ ID NO: 175 (Kabat), or 176 (Chothia);a HCDR3 comprising SEQ ID NO: 177; a LCDR1 comprising SEQ ID NO: 178; aLCDR2 comprising SEQ ID NO: 179; and a LCDR3 comprising SEQ ID NO: 180;

(m) a HCDR1 comprising SEQ ID NO: 181 (Kabat), 182 (Chothia), or 183(extended); a HCDR2 comprising SEQ ID NO: 184 (Kabat), or 185 (Chothia);a HCDR3 comprising SEQ ID NO: 186; a LCDR1 comprising SEQ ID NO: 187; aLCDR2 comprising SEQ ID NO: 188; and a LCDR3 comprising SEQ ID NO: 189;

(n) a HCDR1 comprising SEQ ID NO: 190 (Kabat), 191 (Chothia), or 192(extended); a HCDR2 comprising SEQ ID NO: 193 (Kabat), or 194 (Chothia);a HCDR3 comprising SEQ ID NO: 195; a LCDR1 comprising SEQ ID NO: 196; aLCDR2 comprising SEQ ID NO: 197; and a LCDR3 comprising SEQ ID NO: 198;

(o) a HCDR1 comprising SEQ ID NO: 199 (Kabat), 200 (Chothia), or 201(extended); a HCDR2 comprising SEQ ID NO: 202 (Kabat), or 203 (Chothia);a HCDR3 comprising SEQ ID NO: 204; a LCDR1 comprising SEQ ID NO: 205; aLCDR2 comprising SEQ ID NO: 206; and a LCDR3 comprising SEQ ID NO: 207;

(p) a HCDR1 comprising SEQ ID NO: 208 (Kabat), 209 (Chothia), or 210(extended); a HCDR2 comprising SEQ ID NO: 211 (Kabat), or 212 (Chothia);a HCDR3 comprising SEQ ID NO: 213; a LCDR1 comprising SEQ ID NO: 214; aLCDR2 comprising SEQ ID NO: 215; and a LCDR3 comprising SEQ ID NO: 216;or

(q) a HCDR1 comprising SEQ ID NO: 217; a HCDR2 comprising SEQ ID NO:218; a HCDR3 comprising SEQ ID NO: 219; a LCDR1 comprising SEQ ID NO:220; a LCDR2 comprising SEQ ID NO: 221; and a LCDR3 comprising SEQ IDNO: 222.

12. The antibody or antigen-binding portion of any one of paragraphs1-10, comprising:(a) a heavy chain variable domain (V_(H)) comprising:

-   -   i) an HCDR3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,        23, 25, 27, 29, or 71 as shown in Table 7; or    -   ii) HCDR1-3 in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21,        23, 25, 27, 29, or 71 as shown in Table 7; or        (b) a light chain variable domain (V_(L)) comprising:    -   i) an LCDR3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,        22, 24, 26, 28, 30, or 72 as shown in Table 7; or    -   ii) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,        22, 24, 26, 28, 30, or 72 as shown in Table 7.        13. An isolated antibody or an antigen-binding portion thereof        that specifically binds human interleukin-2 (hIL-2), comprising        a heavy chain variable domain (V_(H)) comprising:    -   a) an HCDR3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,        23, 25, 27, 29, 31, or 71 as shown in Table 7;    -   b) HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,        23, 25, 27, 29, 31, or 71 as shown in Table 7; or    -   c) the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,        15, 17, 19, 21, 23, 25, 27, 29, 31, or 71.        14. An isolated antibody or an antigen-binding portion thereof        that specifically binds human interleukin-2 (hIL-2), comprising        a light chain variable domain (V_(L)) comprising:    -   a) an LCDR3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,        22, 24, 26, 28, 30, 32, or 72 as shown in Table 7;    -   b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,        24, 26, 28, 30, 32, or 72 as shown in Table 7; or    -   c) the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,        16, 18, 20, 22, 24, 26, 28, 30, 32, or 72.        15. An isolated antibody or an antigen-binding portion thereof        that specifically binds human IL-2 (hIL-2), wherein the antibody        comprises the HCDR1-3 and LCDR1-3 amino acid sequences in:

SEQ ID NOs: 1 and 2,

SEQ ID NOs: 3 and 4,

SEQ ID NOs: 5 and 6,

SEQ ID NOs: 7 and 8,

SEQ ID NOs: 9 and 10,

SEQ ID NOs: 11 and 12,

SEQ ID NOs: 13 and 14,

SEQ ID NOs: 15 and 16,

SEQ ID NOs: 17 and 18,

SEQ ID NOs: 19 and 20,

SEQ ID NOs: 21 and 22,

SEQ ID NOs: 23 and 24,

SEQ ID NOs: 25 and 26,

SEQ ID NOs: 27 and 28,

SEQ ID NOs: 29 and 30,

SEQ ID NOs: 31 and 32, or

SEQ ID NOs: 71 and 72,

respectively, as shown in Table 7.16. The isolated antibody or antigen-binding portion of paragraph 15,wherein the antibody comprises the amino acid sequences of

SEQ ID NOs: 1 and 2,

SEQ ID NOs: 3 and 4,

SEQ ID NOs: 5 and 6,

SEQ ID NOs: 7 and 8,

SEQ ID NOs: 9 and 10,

SEQ ID NOs: 11 and 12,

SEQ ID NOs: 13 and 14,

SEQ ID NOs: 15 and 16,

SEQ ID NOs: 17 and 18,

SEQ ID NOs: 19 and 20,

SEQ ID NOs: 21 and 22,

SEQ ID NOs: 23 and 24,

SEQ ID NOs: 25 and 26,

SEQ ID NOs: 27 and 28,

SEQ ID NOs: 29 and 30,

SEQ ID NOs: 31 and 32, or

SEQ ID NOs: 71 and 72.

17. The antibody or antigen-binding portion of paragraph 15 or 16,wherein the antibody is an IgG antibody.18. The antibody or antigen-binding portion of paragraph 17, furthercomprising a heavy chain constant region comprising the amino acidsequence of SEQ ID NO: 33.19. The antibody or antigen-binding portion of paragraph 17, furthercomprising a heavy chain constant region comprising the amino acidsequence of SEQ ID NO: 33 without the C-terminal lysine.20. The antibody or antigen-binding portion of paragraph 18 or 19,further comprising a light chain constant region comprising the aminoacid sequence of SEQ ID NO: 34 or 35.21. An isolated nucleic acid encoding the heavy chain, the light chain,or both, of the antibody or antigen-binding portion of any one ofparagraphs 11-20.22. An isolated nucleic acid encoding the heavy chain, the light chain,or both, of an antibody or an antigen-binding portion thereof thatspecifically binds human interleukin-2 (hIL-2), wherein said nucleicacid comprises:

-   -   a) the nucleotide sequence of SEQ ID NO: 36, 38, 40, 42, 44, 46,        48, 50, 52, 54, 56, 58, 60, 62, 64, or 66;    -   b) the nucleotide sequence of SEQ ID NO: 37, 39, 41, 43, 45, 47,        49, 51, 53, 55, 57, 59, 61, 63, 65, or 67; or    -   c) one of the following nucleotide sequence pairs:        -   SEQ ID NOs: 36 and 37,        -   SEQ ID NOs: 38 and 39,        -   SEQ ID NOs: 40 and 41,        -   SEQ ID NOs: 42 and 43,        -   SEQ ID NOs: 44 and 45,        -   SEQ ID NOs: 46 and 47,        -   SEQ ID NOs: 48 and 49,        -   SEQ ID NOs: 50 and 51,        -   SEQ ID NOs: 52 and 53,        -   SEQ ID NOs: 54 and 55,        -   SEQ ID NOs: 56 and 57,        -   SEQ ID NOs: 58 and 59,        -   SEQ ID NOs: 60 and 61,        -   SEQ ID NOs: 62 and 63,        -   SEQ ID NOs: 64 and 65, or        -   SEQ ID NOs: 66 and 67.            23. A vector comprising the nucleic acid of paragraph 21 or            22.            24. A host cell comprising the nucleic acid of paragraph 21            or 22 or the vector of paragraph 23.            25. The host cell of paragraph 24, wherein the cell is a            mammalian cell.            26. A method of producing an antibody or an antigen-binding            portion thereof that specifically binds human interleukin-2            (hIL-2), said method comprising:

a) culturing the host cell of paragraph 24 or 25 under conditions thatallow said antibody or antigen-binding portion to be expressed, whereinthe host cell comprises nucleotide sequences coding the heavy chain andlight chain of the antibody or antigen-binding portion, and

b) isolating said antibody or antigen-binding portion from the culture.

27. An isolated antibody or an antigen-binding portion thereof thatspecifically binds human interleukin-2 (hIL-2), wherein the antibodycompetes for binding to hIL-2 with, or binds to the same epitope as, theantibody or antigen-binding portion of paragraph 16.28. The antibody or antigen-binding portion of paragraph 27, wherein theantibody comprises a V_(H) amino acid sequence and a V_(L) amino acidsequence that are at least 95% identical to the following amino acidsequences, respectively:

SEQ ID NOs: 1 and 2,

SEQ ID NOs: 3 and 4,

SEQ ID NOs: 5 and 6,

SEQ ID NOs: 7 and 8,

SEQ ID NOs: 9 and 10,

SEQ ID NOs: 11 and 12,

SEQ ID NOs: 13 and 14,

SEQ ID NOs: 15 and 16,

SEQ ID NOs: 17 and 18,

SEQ ID NOs: 19 and 20,

SEQ ID NOs: 21 and 22,

SEQ ID NOs: 23 and 24,

SEQ ID NOs: 25 and 26,

SEQ ID NOs: 27 and 28,

SEQ ID NOs: 29 and 30, or

SEQ ID NOs: 31 and 32.

29. A pharmaceutical composition comprising the antibody orantigen-binding portion of any one of paragraphs 1-20, 27, and 28, and apharmaceutically acceptable carrier or excipient.30. A method for treating an inflammatory condition in a human subject,comprising administering to the subject an effective amount of theantibody or antigen-binding portion of any one of paragraphs 1-20, 27,and 28, or the pharmaceutical composition of paragraph 29.31. A method for inducing immunosuppression in a human subject in needthereof, comprising administering to the subject an effective amount ofthe antibody or antigen-binding portion of any one of paragraphs 1-20,27, and 28, or the pharmaceutical composition of paragraph 29.32. An antibody or antigen-binding portion of any one of paragraphs1-20, 27, and 28, or a pharmaceutical composition of paragraph 29, foruse in treating a human subject having an inflammatory condition or inneed of immunosuppression.33. Use of an antibody or antigen-binding portion of any one ofparagraphs 1-20, 27, and 28 in the manufacture of a medicament fortreating an inflammatory condition or inducing immunosuppression in ahuman subject in need thereof.34. The method of paragraph 30 or 31, the antibody, portion orpharmaceutical composition of paragraph 32, or the use of paragraph 33,wherein the subject has an autoimmune disease or graft versus hostdisease.35. The method of paragraph 30 or 31, the antibody, portion orpharmaceutical composition of paragraph 32, or the use of paragraph 33,wherein the antibody or portion is administered in complex with IL-2.36. The method of paragraph 30 or 31, the antibody, portion orpharmaceutical composition of paragraph 32, or the use of paragraph 33,wherein the subject has diabetes mellitus.37. The method of paragraph 30 or 31, the antibody, portion orpharmaceutical composition of paragraph 32, or the use of paragraph 33,wherein the subject has Type I diabetes mellitus.38. A composition comprising the antibody or antigen-binding portion ofany one of paragraphs 1-20, 27, and 27 complexed with human IL-2.39. A pharmaceutical composition comprising the antibody orantigen-binding portion of any one of paragraphs 1-20, 27, and 28,complexed with human IL-2, and a pharmaceutically acceptable carrier orexcipient.40. A method for treating an inflammatory condition in a human subject,comprising administering to the subject an effective amount of theantibody or antigen-binding portion of any one of paragraphs 1-20, 27,and 28 complexed with human IL-2, the pharmaceutical composition ofparagraph 29, the composition of paragraph 38, or the pharmaceuticalcomposition of paragraph 39.41. An isolated antibody F5.1.11.02 or an antigen-binding portionthereof that specifically binds human interleukin-2 (hIL-2), wherein theantibody comprises: a heavy chain variable domain (VH) comprising theamino acid sequence encoded by the cDNA insert of the plasmid depositedunder ATCC accession number and/or a light chain variable domain (VL)comprising the amino acid sequence encoded by the cDNA insert of theplasmid deposited under ATCC accession number

TABLE 7 Sequence Listinq TableKabat CDR amino acid sequences are in bold. ChothiaCDR amino acid sequences are underlined. ExtendedCDR amino acid sequences include both underlinedand bold amino acid residues. F4.7.6 VH SEQ ID EVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS NO: 1 WIRQHPGKGLEWIGY IYYSGSTYYNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WG QGTLVTVSSF4.7.6 VL SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ NO: 2QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASL TISGLKTEDEADYYC QSYDSSTVVFGGGTKLTVL F4.7.8 VH SEQ ID  QVQLVQSGAEVKKPGASVKVSCKASGYTFTGY YMHW NO: 3VRQAPGQGLEWMGW INPNSGGTN YAQKFQGRVTMT RDTSISTAYMELSRLRSDDTAVYYCAR DLTFDYWGQG TLVTVSS F4.7.8 VL SEQ ID  AIQLTQSPSSLSASVGDRVTITC QASQDIFNLLN WYRQNO: 4 KPGKAPDLLVY RASNLET GVPSRFSGSGSGTDFTFTIS SLQPEDVGTYYC QQSANLPLTFGGGTKVEIK F5.1.11 SEQ ID  QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VHNO: 5 WIRQHPGKGLEWIGY IYYSGSTY YNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCARTPTVTGDWFDP WG RGTLVTVSS F5.1.11 SEQ ID  NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQ WYQ VL NO: 6 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDSSNVV FGGGTKLTVL F5.1.9 VH SEQ ID EVQLVESGPGLVKPSETLSLTCAVSGYSISSGY YWGWI NO: 7 RQPPGKGLEWIGS IYHSGSTYYNPSLKSRVTISVDTSK NQFSLKLSSVTAADTAVYYCAR EAYSDRAFDI WGQGT MVTVSSF5.1.9 VL SEQ ID  NFMLTQPHSVSESPGKTITISC TRSSGSIASDYVQ WYQ NO: 8QRPGSSPSTVIY ADNQRPS EVPDRFSGSIDSSSNSASL TISGLMTEDEADYYC QSYDSNIVIFGGGTKLTVL F4.7.062 SEQ ID  QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWS VHNO: 9 WIRQHPGKGLEWIGY IYYSGSTY YNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCARTPTVTGDWFDP WG QGTLVTVSS F4.7.062 SEQ ID  NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQ WYQ VL NO: 10 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDSSNVV FGGGTKLTVL F5.1.11.01 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 11 WIRQHPGKGLEWIGYIYKSGSAY YSPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.01 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 12 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASL TISGLKTEDEADYYCQSYDTRDVV FGGGTKLTVL F5.1.11.02 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 13 WIRQHPGKGLEWIGYIYKSGSAY YSPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.02 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 14 QRPGSSPTTVIY EDNQRPS GVPDRFSGSidSSSNSASL TISGLKTEDEADYYCQTYDSIDVY FGGGTKLTVL F5.1.11.03 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 15 WIRQHPGKGLEWIGYIYKSGSAY YSPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.03 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 16 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSAST ISGLKTEDEADYYC QSYDTLNVYFGGGTKLTVL F5.1.11.04 SEQ ID  QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VHNO: 17 WIRQHPGKGLEWIGY IYYSGSNY WNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCARTPTVTGDWFDP WG RGTLVTVSS F5.1.11.04 SEQ ID  NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQ WYQ VL NO: 18 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDTRDVV FGGGTKLTVL F5.1.11.05 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 19 WIRQHPGKGLEWIGYIYYSGSNY WNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.05 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 20 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASL TISGLKTEDEADYYCQTYDSIDVY FGGGTKLTVL F5.1.11.06 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 21 WIRQHPGKGLEWIGYIYYSGSNY WNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.06 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 22 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASL TISGLKTEDEADYYCQSYDTLNVY FGGGTKLTVL F5.1.11.07 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 23 WIRQHPGKGLEWIGYIYKSGSNY WNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.07 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 24 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASL TISGLKTEDEADYYCQSYDTRDVV FGGGTKLTVL F5.1.11.08 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 25 WIRQHPGKGLEWIGYIYKSGSNY WNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.08 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQ VLNO: 26 QRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASL TISGLKTEDEADYYCQTYDSIDVY FGGGTKLTVL F5.1.11.09 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGY YWS VH NO: 27 WIRQHPGKGLEWIGYIYKSGSNY WNPSLKSRVTISVDT SKNQFSLKLSSVTAADTAVYYCAR TPTVTGDWFDP WGRGTLVTVSS F5.1.11.09 SEQ ID  NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ VLNO: 28 WYQQRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSN SASLTISGLKTEDEADYYCQSYDTLNVY FGGGTKLTVL F5.1.9.5 SEQ ID  EVQLVESGPGLVKPSETLSLTCAVSGYSISSGYYWGWI VH NO: 29 RQPPGKGLEWIGL SYHTRSTY YDPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR EAYSDRAFDI WGQGT MVTVSS F5.1.9.5 SEQ ID NFMLTQPHSVSESPGKTITISC TRSSGSIASDYVQ WYQ VL NO: 30 QRPGSSPSTVIY ADNQRPSEVPDRFSGSIDSSSNSASL TISGLMTEDEADYYC QSYDSNIVI FGGGTKLTVL d1C7 VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY AISWV NO: 31 RQAPGQGLEWMGG IIPIFGTANYAQKFQGRVTITADES TSTAYMELSSLRSEDTAVYYCAR VDRYYNWNYFLGSF DY WGQGTLVTVSSd1C7 VL SEQ ID  SYVLTQPPSVSVAPGKTARITC GGNNIRSKSVH WYQQ NO: 32KPGQAPVVVIY YDSDRPS GIPERISGSNSGNTATLTISR VEAGDEADYFC QVWDSSSDHHVFGGGTKLTVL HEAVY C SEQ ID  ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT NO: 33VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKLIGHT C SEQ ID  RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK KAPPA NO: 34VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGECLIGHT C SEQ ID  GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT LAMBDA NO: 35VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP EQWKSHRSYSCQVTHEGSTVEKTVAPTECSF4.7.6 VH SEQ ID  GAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGGT NO: 36GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTA CTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTC TCGAGC F4.7.6 VL SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT NO: 37CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGC AGCACCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA F4.7.8 VH  SEQ ID  CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAA NO: 38GAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGA TGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACCTAACGTTTGACTACTGGGGCCAGG GAACCCTGGTCACCGTCTCGAGC F4.7.8 VLSEQ ID  GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCT NO: 39GCGTCTGTAGGAGACAGGGTCACCATCACTTGCCAGGCGAGTCAGGACATTTTCAACCTCTTAAATTGGTATAGGCAGAAACCAGGGAAAGCCCCTGACCTCCTGGTCTACCGCGCTTCCAATTTGGAGACAGGGGTCCCATCCAGGTTCAGTGGAAGTGGGTCTGGGACAGACTTTACTTTCACCATTAGTAGCCTGCAGCCTGAAGATGTTGGAACCTATTATTGTCAACAGAGTGCTAATCTCCCCCTCAC TTTCGGCGGAGGGACCAAGGTGGAGATCAAAF5.1.11 SEQ ID  CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 40GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTA CTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGTC TCGAGC F5.1.11 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 41CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGC AGCAATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA F5.1.9 VH SEQ ID  GAGGTGCAGCTGGTGGAGTCGGGCCCAGGACTGGT NO: 42GAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCAGCAGTGGTTACTACTGGGG CTGGATCCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATCATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGAGAGGCCTACTCCGATCGGGCTTTTGATA TCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGCF5.1.9 VL SEQ ID  AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT NO: 43CTCCGGGGAAGACGATAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCGACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCAGCACTGTGATCTATGCGGATAACCAAAGACCCTCTGAAGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGATGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAACATCGTGATATTCGGCGGAGGGACCAAGCTGACC GTCCTA F4.7.062  SEQ ID CAGGTGCAGCTACAGGAGTCGGGCCCAGGACTGGT VHGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGT NO: 44CTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTA CTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTC TCGAGC F4.7.062 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 45CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGC AGCAATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA F5.1.11.01 SEQ ID  CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 46GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATATATCTATAAGAGTGGGAGCGCGTACTACAGCCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATT ACTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGT CTCCTCA F5.1.11.01 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 47CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCAGGGATGTCGTATTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.02 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 48GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATATATCTATAAGAGTGGGAGCGCGTACTACAGCCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATT ACTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGT CTCCTCA F5.1.11.02 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 49CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGACTTATGACAGCATCGATGTGTATTTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.03 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 50GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATATATCTATAAGAGTGGGAGCGCGTACTACAGCCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATT ACTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGT CTCCTCA F5.1.11.03 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 51CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCCTTAATGTGTATTTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.04 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 52GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATTTATTACAGCGGGAGCAACTACTGGAATCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTA CTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGTC TCCTCA F5.1.11.04 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 53CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCAGGGATGTCGTATTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.05 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 54GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATTTATTACAGCGGGAGCAACTACTGGAATCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTA CTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGTC TCCTCA F5.1.11.05 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 55CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGACTTATGACAGCATCGATGTGTATTTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.06 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 56GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATTTATTACAGCGGGAGCAACTACTGGAATCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTA CTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGTC TCCTCA F5.1.11.06 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 57CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCCTTAATGTGTATTTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.07 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 58GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATAAGAGCGGGAGCAACTACTGGAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATT ACTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGT CTCCTCA F5.1.11.07 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 59CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCAGGGATGTCGTATTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.08 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 60GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATAAGAGCGGGAGCAACTACTGGAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATT ACTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGT CTCCTCA F5.1.11.08 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 61CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGACTTATGACAGCATCGATGTGTATTTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.11.09 SEQ ID CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGT VH NO: 62GAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTG GAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATAAGAGCGGGAGCAACTACTGGAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATT ACTGTGCGAGGACTCCTACGGTGACCGGGGACTGGTTCGACCCCTGGGGCCGTGGCACCCTGGTCACCGT CTCCTCA F5.1.11.09 SEQ ID AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 63CTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCCTTAATGTGTATTTCGGCGGAGGGACCAAGCTGACC GTCCTA F5.1.9.5 SEQ ID GAGGTGCAGCTGGTGGAGTCGGGCCCAGGACTGGT VH NO: 64GAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCAGCAGTGGTTACTACTGGGG CTGGATCCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGTTTGAGCTACCACACTCGTTCTACCTACTACGATCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGAGAGGCCTACTCCGATCGGGCTTTTGATAT CTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAF5.1.9.5 SEQ ID  AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGT VL NO: 65CTCCGGGGAAGACGATAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCGACTATGTGCAGTGGT ACCAGCAGCGCCCGGGCAGTTCCCCCAGCACTGTGATCTATGCGGATAACCAAAGACCCTCTGAAGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGATGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAACATCGTGATATTCGGCGGAGGGACCAAGCTGACC GTCCTA d1C7 VH SEQ ID CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAA NO: 66GAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG CTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGG ATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACC GCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTGGACCGGTATTATAACTGGAACTACTTTTTAGGCTCCTTTGACTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCGAGC d1C7 VL SEQ ID AGCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTG NO: 67GCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTAGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCTGTGGTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGAATCTCTGGGTCCAACTCTGGAAACACGGCCACCCTG ACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTTTTGTCAGGTGTGGGATAGTAGTAGTGACCATCATGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA Heavy C SEQ ID GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA NO: 68CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGG CACCGTCAGTCTTCCTCTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Light C SEQ ID CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCG Kappa NO: 69CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTLight C SEQ ID  GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTC Lambda NO: 70CCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGA GCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCA AACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAA GCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA F5.1.11.02 SEQ ID QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWS library VH NO: 71WIRQHPGKGLEWIGYIYKSGSAYYSPSLKSRVTISVDT (ital.SKNQFSLKLSSVTAADTAVYYCARTPTVTGDWFDPWG random- RGTLVTVSS ized except C)F5.1.11.02 SEQ ID  NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQ library VLNO: 72 QRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASL (ital.TISGLKTEDEADYYCQTYDSIDVYFGGGTKLTVL random- ized except C) F4.7.6 SEQ ID SGGYYWS HCDR1 NO: 73 Kabat F4.7.6 SEQ ID  GSISSGGY HCDR1 NO: 74 ChothiaF4.7.6 SEQ ID  GSISSGGYYWS HCDR1 NO: 75 Extended F4.7.6 SEQ ID GYIYYSGSTYYNPSLKSRV HCDR2 NO: 76 Kabat F4.7.6 SEQ ID  IYYSGSTY HCDR2NO: 77 Chothia F4.7.6 SEQ ID  TPTVTGDWFDP HCDR3 NO: 78 F4.7.6 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 79 F4.7.6 SEQ ID  EDNQRPS LCDR2 NO: 80 F4.7.6SEQ ID  QSYDSSTVV LCDR3 NO: 81 F4.7.8 SEQ ID  GYYMH HCDR1 NO: 82 KabatF4.7.8 SEQ ID  YTFTGY HCDR1 NO: 83 Chothia F4.7.8 SEQ ID  YTFTGYYMHHCDR1 NO: 84 Extended F4.7.8 SEQ ID  GWINPNSGGTNYAQKFQGRV HCDR2 NO: 85Kabat F4.7.8 SEQ ID  INPNSGGTN HCDR2 NO: 86 Chothia F4.7.8 SEQ ID DLTFDY HCDR3 NO: 87 F4.7.8 SEQ ID  QASQDIFNLLN LCDR1 NO: 88 F4.7.8SEQ ID  RASNLET LCDR2 NO: 89 F4.7.8 SEQ ID  QQSANLPLT LCDR3 NO: 90F5.1.11 SEQ ID  SGGYYWS HCDR1 NO: 91 Kabat F5.1.11 SEQ ID  GSISSGGYHCDR1 NO: 92 Chothia F5.1.11 SEQ ID  GSISSGGYYWS HCDR1 NO: 93 ExtendedF5.1.11 SEQ ID  GYIYYSGSTYYNPSLKSRV HCDR2 NO: 94 Kabat F5.1.11 SEQ ID IYYSGSTY HCDR2 NO: 95 Chothia F5.1.11 SEQ ID  TPTVTGDWFDP HCDR3 NO: 96F5.1.11 SEQ ID  TRSSGSIASNYVQ LCDR1 NO: 97 F5.1.11 SEQ ID  EDNQRPS LCDR2NO: 98 F5.1.11 SEQ ID  QSYDSSNVV LCDR3 NO: 99 F5.1.9 SEQ ID SGYYWG HCDR1NO: 100 Kabat F5.1.9 SEQ ID YSISSGY HCDR1 NO: 101 Chothia F5.1.9 SEQ IDYSISSGYYWG HCDR1 NO: 102 Extended F5.1.9 SEQ ID GSIYHSGSTYYNPSLKSRVHCDR2 NO: 103 Kabat F5.1.9 SEQ ID IYHSGSTY HCDR2 NO: 104 Chothia F5.1.9SEQ ID EAYSDRAFDI HCDR3 NO: 105 F5.1.9 SEQ ID TRSSGSIASDYVQ LCDR1NO: 106 F5.1.9 SEQ ID ADNQRPS LCDR2 NO: 107 F5.1.9 SEQ ID QSYDSNIVILCDR3 NO: 108 F4.7.062 SEQ ID SGGYYWS HCDR1 NO: 109 Kabat F4.7.062SEQ ID GSISSGGY HCDR1 NO: 110 Chothia F4.7.062 SEQ ID GSISSGGYYWS HCDR1NO: 111 Extended F4.7.062 SEQ ID GYIYYSGSTYYNPSLKSRV HCDR2 NO: 112 KabatF4.7.062 SEQ ID IYYSGSTY HCDR2 NO: 113 Chothia F4.7.062 SEQ IDTPTVTGDWFDP HCDR3 NO: 114 F4.7.062 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 115F4.7.062 SEQ ID EDNQRPS LCDR2 NO: 116 F4.7.062 SEQ ID QSYDSSNVV LCDR3NO: 117 F5.1.11.01 SEQ ID SGGYYWS HCDR1 NO: 118 Kabat F5.1.11.01 SEQ IDGSISSGGY Chothia NO: 119 HCDR1 F5.1.11.01 SEQ ID GSISSGGYYWS HCDR1NO: 120 Extended F5.1.11.01 SEQ ID GYIYKSGSAYYSPSLKSRV HCDR2 NO: 121Kabat F5.1.11.01 SEQ ID IYKSGSAY HCDR2 NO: 122 Chothia F5.1.11.01 SEQ IDTPTVTGDWFDP HCDR3 NO: 123 F5.1.11.01 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 124F5.1.11.01 SEQ ID EDNQRPS LCDR2 NO: 125 F5.1.11.01 SEQ ID QSYDTRDVVLCDR3 NO: 126 F5.1.11.02 SEQ ID SGGYYWS HCDR1 NO: 127 Kabat F5.1.11.02SEQ ID GSISSGGY HCDR1 NO: 128 Chothia F5.1.11.02 SEQ ID GSISSGGYYWSHCDR1 NO: 129 Extended F5.1.11.02 SEQ ID GYIYKSGSAYYSPSLKSRV HCDR2NO: 130 Kabat F5.1.11.02 SEQ ID IYKSGSAY HCDR2 NO: 131 ChothiaF5.1.11.02 SEQ ID TPTVTGDWFDP HCDR3 NO: 132 F5.1.11.02 SEQ IDTRSSGSIASNYVQ LCDR1 NO: 133 F5.1.11.02 SEQ ID EDNQRPS LCDR2 NO: 134F5.1.11.02 SEQ ID QTYDSIDVY LCDR3 NO: 135 F5.1.11.03 SEQ ID SGGYYWSHCDR1 NO: 136 Kabat F5.1.11.03 SEQ ID GSISSGGY HCDR1 NO: 137 ChothiaF5.1.11.03 SEQ ID GSISSGGYYWS HCDR1 NO: 138 Extended F5.1.11.03 SEQ IDGYIYKSGSAYYSPSLKSRV HCDR2 NO: 139 Kabat F5.1.11.03 SEQ ID IYKSGSAY HCDR2NO: 140 Chothia F5.1.11.03 SEQ ID TPTVTGDWFDP HCDR3 NO: 141 F5.1.11.03SEQ ID TRSSGSIASNYVQ LCDR1 NO: 142 F5.1.11.03 SEQ ID EDNQRPS LCDR2NO: 143 F5.1.11.03 SEQ ID QSYDTLNVY LCDR3 NO: 144 F5.1.11.04 SEQ IDSGGYYWS HCDR1 NO: 145 Kabat F5.1.11.04 SEQ ID GSISSGGY HCDR1 NO: 146Chothia F5.1.11.04 SEQ ID GSISSGGYYWS HCDR1 NO: 147 Extended F5.1.11.04SEQ ID GYIYYSGSNYWNPSLKSRV HCDR2 NO: 148 Kabat F5.1.11.04 SEQ IDIYYSGSNY HCDR2 NO: 149 Chothia F5.1.11.04 SEQ ID TPTVTGDWFDP HCDR3NO: 150 F5.1.11.04 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 151 F5.1.11.04 SEQ IDEDNQRPS LCDR2 NO: 152 F5.1.11.04 SEQ ID QSYDTRDVV LCDR3 NO: 153F5.1.11.05 SEQ ID SGGYYWS HCDR1 NO: 154 Kabat F5.1.11.05 SEQ ID GSISSGGYHCDR1 NO: 155 Chothia F5.1.11.05 SEQ ID GSISSGGYYWS HCDR1 NO: 156Extended F5.1.11.05 SEQ ID GYIYYSGSNYWNPSLKSRV HCDR2 NO: 157 KabatF5.1.11.05 SEQ ID IYYSGSNY HCDR2 NO: 158 Chothia F5.1.11.05 SEQ IDTPTVTGDWFDP HCDR3 NO: 159 F5.1.11.05 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 160F5.1.11.05 SEQ ID EDNQRPS LCDR2 NO: 161 F5.1.11.05 SEQ ID QTYDSIDVYLCDR3 NO: 162 F5.1.11.06 SEQ ID SGGYYWS HCDR1 NO: 163 Kabat F5.1.11.06SEQ ID GSISSGGY HCDR1 NO: 164 Chothia F5.1.11.06 SEQ ID GSISSGGYYWSHCDR1 NO: 165 Extended F5.1.11.06 SEQ ID GYIYYSGSNYWNPSLKSRV HCDR2NO: 166 Kabat F5.1.11.06 SEQ ID IYYSGSNY HCDR2 NO: 167 ChothiaF5.1.11.06 SEQ ID TPTVTGDWFDP HCDR3 NO: 168 F5.1.11.06 SEQ IDTRSSGSIASNYVQ LCDR1 NO: 169 F5.1.11.06 SEQ ID EDNQRPS LCDR2 NO: 170F5.1.11.06 SEQ ID QSYDTLNVY LCDR3 NO: 171 F5.1.11.07 SEQ ID SGGYYWSHCDR1 NO: 172 Kabat F5.1.11.07 SEQ ID GSISSGGY HCDR1 NO: 173 ChothiaF5.1.11.07 SEQ ID GSISSGGYYWS HCDR1 NO: 174 Extended F5.1.11.07 SEQ IDGYIYKSGSNYWNPSLKSRV HCDR2 NO: 175 Kabat F5.1.11.07 SEQ ID IYKSGSNY HCDR2NO: 176 Chothia F5.1.11.07 SEQ ID TPTVTGDWFDP HCDR3 NO: 177 F5.1.11.07SEQ ID TRSSGSIASNYVQ LCDR1 NO: 178 F5.1.11.07 SEQ ID EDNQRPS LCDR2NO: 179 F5.1.11.07 SEQ ID QSYDTRDVV LCDR3 NO: 180 F5.1.11.08 SEQ IDSGGYYWS HCDR1 NO: 181 Kabat F5.1.11.08 SEQ ID GSISSGGY HCDR1 NO: 182Chothia F5.1.11.08 SEQ ID GSISSGGYYWS HCDR1 NO: 183 Extended F5.1.11.08SEQ ID GYIYKSGSNYWNPSLKSRV HCDR2 NO: 184 Kabat F5.1.11.08 SEQ IDIYKSGSNY HCDR2 NO: 185 Chothia F5.1.11.08 SEQ ID TPTVTGDWFDP HCDR3NO: 186 F5.1.11.08 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 187 F5.1.11.08 SEQ IDEDNQRPS LCDR2 NO: 188 F5.1.11.08 SEQ ID QTYDSIDVY LCDR3 NO: 189F5.1.11.09 SEQ ID SGGYYWS HCDR1 NO: 190 Kabat F5.1.11.09 SEQ ID GSISSGGYHCDR1 NO: 191 Chothia F5.1.11.09 SEQ ID GSISSGGYYWS HCDR1 NO: 192Extended F5.1.11.09 SEQ ID GYIYKSGSNYWNPSLKSRV HCDR2 NO: 193 KabatF5.1.11.09 SEQ ID IYKSGSNY HCDR2 NO: 194 Chothia F5.1.11.09 SEQ IDTPTVTGDWFDP HCDR3 NO: 195 F5.1.11.09 SEQ ID TRSSGSIASNYVQ LCDR1 NO: 196F5.1.11.09 SEQ ID EDNQRPS LCDR2 NO: 197 F5.1.11.09 SEQ ID QSYDTLNVYLCDR3 NO: 198 F5.1.9.5 SEQ ID SGYYWG HCDR1 NO: 199 Kabat F5.1.9.5 SEQ IDYSISSGY HCDR1 NO: 200 Chothia F5.1.9.5 SEQ ID YSISSGYYWG HCDR1 NO: 201Extended F5.1.9.5 SEQ ID GLSYHTRSTYYDPSLKSRV HCDR2 NO: 202 KabatF5.1.9.5 SEQ ID SYHTRSTY HCDR2 NO: 203 Chothia F5.1.9.5 SEQ IDEAYSDRAFDI HCDR3 NO: 204 F5.1.9.5 SEQ ID TRSSGSIASDYVQ LCDR1 NO: 205F5.1.9.5 SEQ ID ADNQRPS LCDR2 NO: 206 F5.1.9.5 SEQ ID QSYDSNIVI LCDR3NO: 207 d1C7 SEQ ID SYAIS HCDR1 NO: 208 Kabat d1C7 SEQ ID GTFSSY HCDR1NO: 209 Chothia d1C7 SEQ ID GTFSSYAIS HCDR1 NO: 210 Extended d1C7 SEQ IDGGIIPIFGTANYAQKFQGRV HCDR2 NO: 211 Kabat d1C7 SEQ ID IIPIFGTAN HCDR2NO: 212 Chothia d1C7 SEQ ID VDRYYNWNYFLGSFDY HCDR3 NO: 213 d1C7 SEQ IDGGNNIRSKSVH LCDR1 NO: 214 d1C7 SEQ ID YDSDRPS LCDR2 NO: 215 d1C7 SEQ IDQVWDSSSDHHV LCDR3 NO: 216 F5.1.11.02 SEQ ID SGGYYWS library NO: 217HCDR1 F5.1.11.02 SEQ ID GYIYKSGSAYYSPSLKSRV library NO: 218 HCDR2F5.1.11.02 SEQ ID TPTVTGDWFDP library NO: 219 HCDR3 (ital. random- izedexcept C) F5.1.11.02 SEQ ID TRSSGSIASNYVQ library NO: 220 LCDR1 (ital.random- ized except C) F5.1.11.02 SEQ ID EDNQRPS library NO: 221 LCDR2F5.1.11.02 SEQ ID QTYDSIDVY library NO: 222 LCDR3

TABLE 8 HUMAN ANTI-IL-2 ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NO:)Variable Domains Monoclonal Light Heavy Antibody DNA Protein DNA ProteinF4.7.6 37 2 36 1 F4.7.8 39 4 38 3 F5.1.11 41 6 40 5 F5.1.9 43 8 42 7F4.7.062 45 10 44 9 F5.1.11.01 47 12 46 11 F5.1.11.02 49 14 48 13F5.1.11.03 51 16 50 15 F5.1.11.04 53 18 52 17 F5.1.11.05 55 20 54 19F5.1.11.06 57 22 56 21 F5.1.11.07 59 24 58 23 F5.1.11.08 61 26 60 25F5.1.11.09 63 28 62 27 F5.1.9.5 65 30 64 29 d1C7 67 32 66 31

What is claimed is:
 1. An isolated antibody or an antigen-bindingportion thereof that specifically binds human IL-2 (hIL-2), wherein theantibody binds helices A and C and the B-C loop of hIL-2.
 2. An isolatedantibody or an antigen-binding portion thereof that specifically bindshuman IL-2 (hIL-2), wherein the antibody reduces the binding affinity ofhIL-2 to IL-2Rα by 1 to 199 fold.
 3. The antibody or antigen-bindingportion of claim 2, wherein the antibody reduces the binding affinity ofhIL-2 to IL-2Rα by 10 fold.
 4. An isolated antibody or anantigen-binding portion thereof that specifically binds human IL-2(hIL-2), wherein the antibody: (a) reduces hIL-2 binding to IL-2Rα andIL-2Rβ, and inhibits an activity in CD8⁺ T cells to a higher degree thanin regulatory T (Treg) cells; (b) reduces hIL-2 binding to IL-2Rα andIL-2Rβ, and inhibits STAT5 phosphorylation in CD8⁺ T cells to a higherdegree than in regulatory T (Treg) cells; (c) reduces hIL-2 binding toIL-2Rα and IL-2Rβ, and increases the ratio of regulatory T (Treg) cellsto CD8⁺ or CD4⁺ T cells or to NK cells as measured in a peripheral bloodmononuclear cell (PBMC) culture or reconstitution assay; or (d) reduceshIL-2 binding to IL-2Rα and IL-2Rβ, and increases expression of one ormore of FOXP3, CD25, and Icos in regulatory T (Treg) cells.
 5. Theisolated antibody or antigen-binding portion of claim 1, wherein theantibody or antigen-binding portion has at least one of the followingproperties: a) binding to hIL-2 with an off-rate greater than about4.53×10⁻⁴ s⁻¹; and b) binding to hIL-2 with a K_(D) greater than about1.14×10⁻¹⁰ M.
 6. The antibody or antigen-binding portion of claim 1,wherein the antibody is a human antibody.
 7. An isolated antibody orantigen-binding portion thereof that specifically binds human IL-2, saidantibody comprising: (i) (a) a HCDR1 comprising SEQ ID NO: 73, 74, or75; a HCDR2 comprising SEQ ID NO: 76, or 77; a HCDR3 comprising SEQ IDNO: 78; a LCDR1 comprising SEQ ID NO: 79; a LCDR2 comprising SEQ ID NO:80; and a LCDR3 comprising SEQ ID NO: 81; (b) a HCDR1 comprising SEQ IDNO: 82, 83, or 84; a HCDR2 comprising SEQ ID NO: 85, or 86; a HCDR3comprising SEQ ID NO: 87; a LCDR1 comprising SEQ ID NO: 88; a LCDR2comprising SEQ ID NO: 89; and a LCDR3 comprising SEQ ID NO: 90; (c) aHCDR1 comprising SEQ ID NO: 91, 92, or 93; a HCDR2 comprising SEQ ID NO:94, or 95; a HCDR3 comprising SEQ ID NO: 96; a LCDR1 comprising SEQ IDNO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ IDNO: 99; (d) a HCDR1 comprising SEQ ID NO: 100, 101, or 102; a HCDR2comprising SEQ ID NO: 103, or 104; a HCDR3 comprising SEQ ID NO: 105; aLCDR1 comprising SEQ ID NO: 106; a LCDR2 comprising SEQ ID NO: 107; anda LCDR3 comprising SEQ ID NO: 108; (e) a HCDR1 comprising SEQ ID NO:109, 110, or 111; a HCDR2 comprising SEQ ID NO: 112, or 113; a HCDR3comprising SEQ ID NO: 114; a LCDR1 comprising SEQ ID NO: 115; a LCDR2comprising SEQ ID NO: 116; and a LCDR3 comprising SEQ ID NO: 117; (f) aHCDR1 comprising SEQ ID NO: 118, 119, or 120; a HCDR2 comprising SEQ IDNO: 121, or 122; a HCDR3 comprising SEQ ID NO: 123; a LCDR1 comprisingSEQ ID NO: 124; a LCDR2 comprising SEQ ID NO: 125; and a LCDR3comprising SEQ ID NO: 126; (g) a HCDR1 comprising SEQ ID NO: 127, 128,or 129; a HCDR2 comprising SEQ ID NO: 130, or 131; a HCDR3 comprisingSEQ ID NO: 132; a LCDR1 comprising SEQ ID NO: 133; a LCDR2 comprisingSEQ ID NO: 134; and a LCDR3 comprising SEQ ID NO: 135; (h) a HCDR1comprising SEQ ID NO: 136, 137, or 138; a HCDR2 comprising SEQ ID NO:139, or 140; a HCDR3 comprising SEQ ID NO: 141; a LCDR1 comprising SEQID NO: 142; a LCDR2 comprising SEQ ID NO: 143; and a LCDR3 comprisingSEQ ID NO: 144; (i) a HCDR1 comprising SEQ ID NO: 145, 146, or 147; aHCDR2 comprising SEQ ID NO: 148, or 149; a HCDR3 comprising SEQ ID NO:150; a LCDR1 comprising SEQ ID NO: 151; a LCDR2 comprising SEQ ID NO:152; and a LCDR3 comprising SEQ ID NO: 153; (j) a HCDR1 comprising SEQID NO: 154, 155, or 156; a HCDR2 comprising SEQ ID NO: 157, or 158; aHCDR3 comprising SEQ ID NO: 159; a LCDR1 comprising SEQ ID NO: 160; aLCDR2 comprising SEQ ID NO: 161; and a LCDR3 comprising SEQ ID NO: 162;(k) a HCDR1 comprising SEQ ID NO: 163, 164, or 165; a HCDR2 comprisingSEQ ID NO: 166, or 167; a HCDR3 comprising SEQ ID NO: 168; a LCDR1comprising SEQ ID NO: 169; a LCDR2 comprising SEQ ID NO: 170; and aLCDR3 comprising SEQ ID NO: 171; (l) a HCDR1 comprising SEQ ID NO: 172,173, or 174; a HCDR2 comprising SEQ ID NO: 175, or 176; a HCDR3comprising SEQ ID NO: 177; a LCDR1 comprising SEQ ID NO: 178; a LCDR2comprising SEQ ID NO: 179; and a LCDR3 comprising SEQ ID NO: 180; (m) aHCDR1 comprising SEQ ID NO: 181, 182, or 183; a HCDR2 comprising SEQ IDNO: 184, or 185; a HCDR3 comprising SEQ ID NO: 186; a LCDR1 comprisingSEQ ID NO: 187; a LCDR2 comprising SEQ ID NO: 188; and a LCDR3comprising SEQ ID NO: 189; (n) a HCDR1 comprising SEQ ID NO: 190, 191,or 192; a HCDR2 comprising SEQ ID NO: 193, or 194; a HCDR3 comprisingSEQ ID NO: 195; a LCDR1 comprising SEQ ID NO: 196; a LCDR2 comprisingSEQ ID NO: 197; and a LCDR3 comprising SEQ ID NO: 198; (o) a HCDR1comprising SEQ ID NO: 199, 200, or 201; a HCDR2 comprising SEQ ID NO:202, or 203; a HCDR3 comprising SEQ ID NO: 204; a LCDR1 comprising SEQID NO: 205; a LCDR2 comprising SEQ ID NO: 206; and a LCDR3 comprisingSEQ ID NO: 207; (p) a HCDR1 comprising SEQ ID NO: 208, 209, or 210; aHCDR2 comprising SEQ ID NO: 211, or 212; a HCDR3 comprising SEQ ID NO:213; a LCDR1 comprising SEQ ID NO: 214; a LCDR2 comprising SEQ ID NO:215; and a LCDR3 comprising SEQ ID NO: 216; or (q) a HCDR1 comprisingSEQ ID NO: 217; a HCDR2 comprising SEQ ID NO: 218; a HCDR3 comprisingSEQ ID NO: 219; a LCDR1 comprising SEQ ID NO: 220; a LCDR2 comprisingSEQ ID NO: 221; and a LCDR3 comprising SEQ ID NO: 222; (ii) a heavychain variable domain (V_(H)) comprising: a) an HCDR3 in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 71 as shown inTable 7; or b) HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, or 71 as shown in Table 7; (iii) a light chainvariable domain (V_(L)) comprising: a) an LCDR3 in SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 72 as shown in Table7; or b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, or 72 as shown in Table 7; (iv) a heavy chain variabledomain (V_(H)) comprising: a) an HCDR3 in SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71 as shown in Table 7; b)HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, or 71 as shown in Table 7; or c) the amino acid sequence of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71;(v) a light chain variable domain (V_(L)) comprising: a) an LCDR3 in SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72as shown in Table 7; b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, or 72 as shown in Table 7; or c) theamino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, or 72; (vi) the HCDR1-3 and LCDR1-3 amino acidsequences as shown in: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ IDNOs: 3 and 4, respectively; c) SEQ ID NOs: 5 and 6, respectively; d) SEQID NOs: 7 and 8, respectively; e) SEQ ID NOs: 9 and 10, respectively; f)SEQ ID NOs: 11 and 12, respectively; g) SEQ ID NOs: 13 and 14,respectively; h) SEQ ID NOs: 15 and 16, respectively; i) SEQ ID NOs: 17and 18, respectively; j) SEQ ID NOs: 19 and 20, respectively; k) SEQ IDNOs: 21 and 22, respectively; I) SEQ ID NOs: 23 and 24, respectively; m)SEQ ID NOs: 25 and 26, respectively; n) SEQ ID NOs: 27 and 28,respectively; o) SEQ ID NOs: 29 and 30, respectively; p) SEQ ID NOs: 31and 32, respectively; or q) SEQ ID NOs: 71 and 72, respectively; asshown in Table 7; (vii) the amino acid sequences as shown in: a) SEQ IDNOs: 1 and 2, respectively; b) SEQ ID NOs: 3 and 4, respectively; c) SEQID NOs: 5 and 6, respectively; d) SEQ ID NOs: 7 and 8, respectively; e)SEQ ID NOs: 9 and 10, respectively; f) SEQ ID NOs: 11 and 12,respectively; g) SEQ ID NOs: 13 and 14, respectively; h) SEQ ID NOs: 15and 16, respectively; i) SEQ ID NOs: 17 and 18, respectively; j) SEQ IDNOs: 19 and 20, respectively; k) SEQ ID NOs: 21 and 22, respectively; I)SEQ ID NOs: 23 and 24, respectively; m) SEQ ID NOs: 25 and 26,respectively; n) SEQ ID NOs: 27 and 28, respectively; o) SEQ ID NOs: 29and 30, respectively; p) SEQ ID NOs: 31 and 32, respectively; or q) SEQID NOs: 71 and 72, respectively.
 8. The antibody or antigen-bindingportion of claim 7, wherein the antibody is an IgG antibody.
 9. Theantibody or antigen-binding portion of claim 8, further comprising aheavy chain constant region comprising the amino acid sequence of SEQ IDNO: 33 with or without the C-terminal lysine.
 10. The antibody orantigen-binding portion of claim 7(vii)(g), further comprising a lightchain constant region comprising the amino acid sequence of SEQ ID NO:35.
 11. An isolated nucleic acid encoding the heavy chain, the lightchain, or both, of the antibody or antigen-binding portion of claim 7.12. An isolated nucleic acid encoding the heavy chain, the light chain,or both, of an antibody or an antigen-binding portion thereof thatspecifically binds human interleukin-2 (hIL-2), wherein said nucleicacid comprises: a) the nucleotide sequence of SEQ ID NO: 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, or 66; b) the nucleotidesequence of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, or 67; or c) one of the following nucleotide sequence pairs:SEQ ID NOs: 36 and 37, SEQ ID NOs: 38 and 39, SEQ ID NOs: 40 and 41, SEQID NOs: 42 and 43, SEQ ID NOs: 44 and 45, SEQ ID NOs: 46 and 47, SEQ IDNOs: 48 and 49, SEQ ID NOs: 50 and 51, SEQ ID NOs: 52 and 53, SEQ IDNOs: 54 and 55, SEQ ID NOs: 56 and 57, SEQ ID NOs: 58 and 59, SEQ IDNOs: 60 and 61, SEQ ID NOs: 62 and 63, SEQ ID NOs: 64 and 65, or SEQ IDNOs: 66 and
 67. 13. A vector comprising the nucleic acid of claim 12.14. A host cell comprising the nucleic acid of claim
 13. 15. The hostcell of claim 14, wherein the cell is a mammalian cell.
 16. A method ofproducing an antibody or an antigen-binding portion thereof thatspecifically binds human interleukin-2 (hIL-2), said method comprising:a) culturing the host cell of claim 15 under conditions that allow saidantibody or antigen-binding portion to be expressed, wherein the hostcell comprises nucleotide sequences coding the heavy chain and lightchain of the antibody or antigen-binding portion, and b) isolating saidantibody or antigen-binding portion from the culture.
 17. An isolatedantibody or an antigen-binding portion thereof that specifically bindshuman interleukin-2 (hIL-2), wherein the antibody competes for bindingto hIL-2 with, or binds to the same epitope as, the antibody orantigen-binding portion of claim
 7. 18. The antibody or antigen-bindingportion of claim 17, wherein the antibody comprises a V_(H) amino acidsequence and a V_(L) amino acid sequence that are at least 95% identicalto the amino acid sequences of claim 7(vii)(g), respectively.
 19. Apharmaceutical composition comprising the antibody or antigen-bindingportion of claim 7, and a pharmaceutically acceptable carrier orexcipient.
 20. A method for treating an inflammatory condition in ahuman subject, comprising administering to the subject an effectiveamount of the antibody or antigen-binding portion of claim
 7. 21. Amethod for inducing immunosuppression in a human subject in needthereof, comprising administering to the subject an effective amount ofthe antibody or antigen-binding portion of claim
 7. 22. An antibody orantigen-binding portion of claim 7, for use in treating a human subjecthaving an inflammatory condition or in need of immunosuppression. 23.Use of an antibody or antigen-binding portion of claim 7 in themanufacture of a medicament for treating an inflammatory condition orinducing immunosuppression in a human subject in need thereof.
 24. Themethod of claim 21, wherein the subject has an autoimmune disease orgraft versus host disease.
 25. The method of claim 21, wherein theantibody is administered in complex with IL-2.
 26. The method of claim21, wherein the subject has type 1 diabetes.
 27. A compositioncomprising the antibody or antigen-binding portion thereof of claim 7complexed with human IL-2.
 28. A pharmaceutical composition comprisingthe antibody or antigen-binding portion thereof of claim 7, complexedwith human IL-2, and a pharmaceutically acceptable carrier or excipient.29. A method for treating an inflammatory condition in a human subject,comprising administering to the subject an effective amount of theantibody or antigen-binding portion thereof of claim 7 complexed withhuman IL-2.